JP2023554034A - Exosomes derived from cells treated with endoplasmic reticulum stress inducers and their uses - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing endoplasmic reticulum stress-induced exosomes derived from mesenchymal stem cells, and uses thereof. Specifically, the immunomodulatory factors of the WJ-MSC-derived exosomes of the present invention are increased by treatment with an endoplasmic reticulum stress inducer such as thapsigargin. TSG-primed WJ-MSC-derived exosomes play an important role in regulating T cell proliferation and T helper cell differentiation in vitro and exhibit effective therapeutic effects in murine colitis models. . Additionally, administration of TSG-primed WJ-MSC-derived exosomes improved the inflammatory response and induced polarization of regulatory T cells and M2-macrophages in vivo. Therefore, the present invention can be effectively used as a composition for preventing or treating inflammatory diseases.

Description

This application claims priority to Korean Patent Application No. 10-2020-0174576 filed on December 14, 2020, and the entire specification is a reference of this application.

The present invention relates to the therapeutic use of endoplasmic reticulum stress induced mesenchymal stem cell-derived exosomes.

Mesenchymal stem cells (MSCs) are known to have immunomodulatory properties, which are mediated by immunosuppressive, vascular regulatory and paracrine factor activities. The known paracrine secretome includes the secretion of immunomodulatory cytokines, growth factors and small membrane vesicles. However, direct use of mesenchymal stem cells has problems such as maintaining their phenotypic and functional stability, and high isolation and handling costs.

Among the mesenchymal stem cell secretomes (MSC secretomes), exosomes mediate the transmission of paracrine factors in intercellular communication and play an important role in immune regulation. Exosomes are small membrane lipid vesicles with a size of 30-150 nm that are released into the extracellular space and transfer proteins, mRNA, microRNA (miRNA) or other components to target cells and activate immunoregulatory pathways. promote. In particular, exosomes secreted from mesenchymal stem cells are known to exhibit the therapeutic efficacy of stem cells in regenerative medicine. The functions of exosomes and the elements they convey differ depending on cell type, and cellular stress can affect the elements conveyed by exosomes.

The endoplasmic reticulum plays an important role in coordinating signal transduction pathways to ensure cellular homeostasis, and newly synthesized proteins undergo folding and translocation across the ER membrane. ) to experience. However, when the ER is unable to maintain homeostasis under stressful pathological and physiological conditions, the Unfolded Protein Response (UPR) can be activated and affect cellular function. Long-term ER dysfunction or stress is involved in the pathogenesis of various diseases, including metabolic diseases and inflammation-related diseases. Thapsigargin (TSG) is known to increase cytoplasmic calcium concentration, deplete ER calcium stores, and induce ER stress.

Korean Patent No. 10-1980453 relates to a composition for promoting the production of exosomes derived from stem cells, and includes pioglitazone, metformin, or AICAR (5-Aminoimidazole-4-carboxamide ribonucleoti). de) of stem cell-derived exosomes using Disclosed are compositions and methods for increasing the production and content of proteins and RNA within exosomes. However, no research or description has been disclosed regarding exosomes secreted from stem cells treated with endoplasmic reticulum stress-inducing substances such as thapsigargin.

Kim, W. , Lee, S. K. , Kwon, Y. W. , Chung, S. G. & Kim, S. Pioglitazone-Primed Mesenchymal Stem Cells Stimulate Cell Proliferation, Collagen Synthesis and Matrix Gene Expression in Tenocytes. International journal of molecular sciences 20, doi:10.3390/ijms20030472 (2019). Kim, H. S. et al. Human umbilical cord blood mesenchymal stem cells reduce colitis in mice by activating NOD2 signaling to COX2. Gastroenterology 145, 1392-1403. e1391-1398, doi:10.1053/j. gastro. 2013.08.033 (2013). Kang, J. Y. et al. Xeno-Free Condition Enhances Therapeutic Functions of Human Wharton’s jelly-Derived Mesenchymal Stem Cells against Experiment mental Colitis by Upregulated Indoleamine 2,3-Dioxygenase Activity. J Clin Med 9, doi:10.3390/jcm9092913 (2020).

As a result of our intensive efforts to provide a mesenchymal stem cell secretome as an alternative to treatments that directly use mesenchymal stem cells, we discovered that by inducing stress in the endoplasmic reticulum with endoplasmic reticulum stress inducers such as thapsigargin. The present invention was completed by confirming that exosomes secreted from leaf stem cells are involved in the activation and polarization of T cells and macrophages.

Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases containing exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress-inducing substance, and uses thereof.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which contains exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress-inducing substance.

According to a preferred embodiment of the invention, the endoplasmic reticulum stress inducer consists of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT) and MG132. It may be one or more selected from the group.

According to a preferred embodiment of the present invention, the mesenchymal stem cells include adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tubes, corneal stroma, lungs, The mesenchymal stem cells may be obtained from one or more tissues selected from the group consisting of muscle and fetal liver.

According to a preferred embodiment of the present invention, the size of the exosomes may be 10-200 nm.

According to a preferred embodiment of the present invention, the inflammatory disease is any one selected from the group consisting of acute or chronic inflammatory disease, chronic bronchitis, rhinitis, arthritis, autoimmune disease, transplant rejection and inflammatory bowel disease. There may be one or more.

The present invention also provides an immunomodulatory composition comprising mesenchymal stem cell-derived exosomes treated with an endoplasmic reticulum stress inducer.

According to a preferred embodiment of the invention, the immunomodulation increases the levels of anti-inflammatory cytokines, regulatory T cells and M2-type macrophages. and pro-inflammatory cytokines (pro-inflammatory cytokines).
It may also be immunomodulation that reduces the levels of cytokines, helper T cells, and M1-type macrophages.

The present invention also provides a method for promoting the production of mesenchymal stem cell-derived exosomes, which includes the step of treating mesenchymal stem cells with an endoplasmic reticulum stress-inducing substance.

The present invention also provides a method for treating an inflammatory disease, comprising administering to a patient with an inflammatory disease a composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

The present invention also provides the use of a composition comprising mesenchymal stem cell-derived exosomes treated with an endoplasmic reticulum stress inducer for use in the prevention or treatment of inflammatory diseases.

Exosomes derived from mesenchymal stem cells (MSCs) have a lower content of membrane-bound proteins, including tetraspanins (CD81, CD63, CD9), heat shock proteins (HSP60, HSP70, HSP90) and TSG 101, and therefore are more sensitive than mother MSCs. It is known that it has low immunogenicity and does not cause rejection due to activation of alloimmune responses at MHC mismatched receptors.

From this, the present inventors used human Wharton cells treated with thapsigargin (TSG), an endoplasmic reticulum (ER) stress inducer, to apply MSC-derived exosomes to the treatment of immune-mediated inflammatory diseases. We confirmed that exosomes (TSG-Exo) secreted from 's jelly-derived MSCs (WJ-MSCs) have improved immunomodulatory properties.

When stimulating WJ-MSCs with TSG, the secretion of exosomes is increased, and the yield and expression of immunomodulatory and anti-inflammatory factors such as IL-10, COX2 or IDO (Indoleamine 2,3-dioxygenase) are increased; Expression levels of pro-inflammatory factors (IFNγ, TNFα or IL-1β) were reduced or remained similar. The above results were similarly confirmed with TSG-Exo, which suggested that TSG-induced exosome release has a secretory mechanism different from inflammation or senescence-associated secretory mechanism (SASP).

Furthermore, although TSG-Exo is derived from human MSCs, no clear immune rejection reaction was observed when it was administered to a mouse colitis model. This meant that it would be advantageous for future clinical applications. TSG-Exo substantially alleviated colitis by reducing the inflammatory response and maintaining intestinal barrier integrity in a murine colitis model. A significant increase in Tregs and M2-type macrophages in the colon of colitis mice treated with TSG-Exo suggests an anti-inflammatory effect of TSG-Exo. Also, Con
When treated with A-stimulated MNCs (mononuclear cells), similar expression occurred in exosomes and exosome-treated MNCs as the anti-inflammatory factors of exosomes were efficiently delivered to MNCs. Since TSG-Exo showed better regulation of T cell/macrophage polarization than control exosomes, the secretome of exosomes can be a reliable indicator for predicting the therapeutic efficacy of exosomes.

TSG-Exo significantly improved the proliferation and Th1 and Th17 differentiation suppressive effects of human peripheral blood-derived T cells, whereas regulatory T cells and M2-type macrophages were found to be more abundant than in the exosome-treated control group. there were. This suggests that intestinal Tregs and macrophages may be mediators for the anti-inflammatory effects of MSC exosomes. Upregulation of Tregs and M2-type macrophages maintained intestinal homeostasis as suggested by disease activity scores, histological scores and decreased neutrophil activity.

Such data imply that TSG-treated MSCs or TSG-Exo were sufficient to induce exosome release of WJ-MSCs and promoted the immunomodulatory properties of TSG-Exo. Demonstrates the ability to provide new therapeutic methods for treatment.

Therefore, the present invention can provide a pharmaceutical composition for preventing or treating inflammatory diseases, which includes exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress-inducing substance.

According to a preferred embodiment of the invention, the endoplasmic reticulum stress inducer consists of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT) and MG132. It may be one or more selected from the group.

According to a preferred embodiment of the present invention, the mesenchymal stem cells include adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tubes, corneal stroma, lungs, The mesenchymal stem cells may be obtained from one or more tissues selected from the group consisting of muscle and fetal liver. Preferably, the mesenchymal stem cells may be mesenchymal stem cells obtained from Wharton's jelly.

According to a preferred embodiment of the present invention, the size of the exosomes may be 10-200 nm. Preferably, the size of the exosome may be 30 to 150 nm, more preferably 50 to 120 nm.

According to a preferred embodiment of the invention, the inflammatory disease is any one selected from the group consisting of acute or chronic inflammatory disease, chronic bronchitis, rhinitis, arthritis, autoimmune disease, transplant rejection and inflammatory bowel disease. There may be one or more. Preferably, the inflammatory disease may be an inflammatory bowel disease, and the inflammatory bowel disease may be ulcerative colitis, Crohn's disease or Behcet's disease.

The present invention also provides an immunomodulatory composition comprising mesenchymal stem cell-derived exosomes treated with an endoplasmic reticulum stress inducer.

According to a preferred embodiment of the invention, the immunomodulation increases the levels of anti-inflammatory cytokines, regulatory T cells and M2-type macrophages. and pro-inflammatory cytokines (pro-inflammatory cytokines).
It may also be immunomodulation that reduces the levels of cytokines, helper T cells, and M1-type macrophages.

The present invention can also provide a method for promoting the production of mesenchymal stem cell-derived exosomes, which includes the step of treating mesenchymal stem cells with an endoplasmic reticulum stress-inducing substance.

The production promotion may mean promotion of the number of exosomes and the production of exosome proteins and mRNA.

The present invention can also provide a method for treating an inflammatory disease, comprising administering to a patient with an inflammatory disease a composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer. .

According to a preferred embodiment of the invention, the endoplasmic reticulum stress inducer consists of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT) and MG132. It may be one or more selected from the group.

According to a preferred embodiment of the present invention, the mesenchymal stem cells include adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tubes, corneal stroma, lungs, The mesenchymal stem cells may be obtained from one or more tissues selected from the group consisting of muscle and fetal liver. Preferably, the mesenchymal stem cells may be mesenchymal stem cells obtained from Wharton's jelly.

According to a preferred embodiment of the present invention, the size of the exosomes may be 10-200 nm. Preferably, the size of the exosome may be 30 to 150 nm, more preferably 50 to 120 nm.

According to a preferred embodiment of the invention, the inflammatory disease is any one selected from the group consisting of acute or chronic inflammatory disease, chronic bronchitis, rhinitis, arthritis, autoimmune disease, transplant rejection and inflammatory bowel disease. There may be one or more. Preferably, the inflammatory disease may be an inflammatory bowel disease, and the inflammatory bowel disease may be ulcerative colitis, Crohn's disease or Behcet's disease.

The present invention can also provide the use of a composition comprising mesenchymal stem cell-derived exosomes treated with an endoplasmic reticulum stress inducer for use in the prevention or treatment of inflammatory diseases.

According to a preferred embodiment of the invention, the endoplasmic reticulum stress inducer consists of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT) and MG132. It may be one or more selected from the group.

According to a preferred embodiment of the present invention, the mesenchymal stem cells include adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tubes, corneal stroma, lungs, The mesenchymal stem cells may be obtained from one or more tissues selected from the group consisting of muscle and fetal liver. Preferably, the mesenchymal stem cells may be mesenchymal stem cells obtained from Wharton's jelly.

According to a preferred embodiment of the present invention, the size of the exosomes may be 10-200 nm. Preferably, the size of the exosome may be 30 to 150 nm, more preferably 50 to 120 nm.

According to a preferred embodiment of the invention, the inflammatory disease is any one selected from the group consisting of acute or chronic inflammatory disease, chronic bronchitis, rhinitis, arthritis, autoimmune disease, transplant rejection and inflammatory bowel disease. There may be one or more. Preferably, the inflammatory disease may be an inflammatory bowel disease, and the inflammatory bowel disease may be ulcerative colitis, Crohn's disease or Behcet's disease.

Hereinafter, the definitions of terms used in this specification are as follows.

In the present invention, "prevention" refers to all actions that suppress inflammatory diseases or delay their onset.

"Improvement" or "treatment" of the present invention refers to improvements in parameters related to inflammatory diseases, such as improvement in the severity of symptoms, by mesenchymal stem cell-derived exosomes treated with the endoplasmic reticulum stress-inducing substance of the present invention. It means any action that is beneficial.

The pharmaceutical compositions of the present invention may be in a variety of parenteral dosage forms. When formulating the composition, one or more buffering agents (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, etc. They can be prepared using agents, binders, adjuvants (eg aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like. As non-aqueous and suspending solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As bases for suppositories, witepsol, macrogol, tween 61, cacao butter, lauric butter, glycerol, gelatin, etc. can be used.

The composition of the present invention can be administered parenterally, and is known in the art in the form of an injection that can be administered intraperitoneally, rectally, intravenously, intramuscularly, subcutaneously, intrauterinely, or intracerebrovascularly. It can be formulated into a dosage form by the following method.

In the case of injections, they must be sterilized and must be protected from contamination by microorganisms such as bacteria and fungi. For injectables, examples of suitable carriers include solvents or dispersions including, but not limited to, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol), mixtures thereof, and/or vegetable oils. It may be a medium. More preferably, suitable carriers include Hank's solution, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. A tonic solution or the like can be used. In order to protect the injection from microbial contamination, it may further contain various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In addition, the injection may often further contain an isotonic agent such as sugar or sodium chloride.

Compositions of the invention are administered in a pharmaceutically effective amount. A pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment; can be determined by factors including drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The compositions of the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single or multiple doses. can do. That is, the total effective amount of the compositions of the invention can be administered to a patient in a single dose or in a fractionated treatment protocol administered over time in multiple doses. ). Considering all of the above factors, it is important to administer an amount that will provide the maximum effect with the minimum amount without side effects, and this can be readily determined by one skilled in the art.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage is preferably 0.01 to 50 mg per kg of body weight per day, more preferably 0.01 to 50 mg per kg of body weight per day, based on exosomes derived from mesenchymal stem cells treated with endoplasmic reticulum stress-inducing substances during parenteral administration. When administered orally, it is preferably administered in an amount of 1 to 30 mg per kg of body weight per day based on the mesenchymal stem cell-derived exosomes treated with the endoplasmic reticulum stress-inducing substance of the present invention. 0.01 to 100 mg, more preferably 0.01 to 10
It can be administered in one to several divided doses, such that the dose is administered in mg doses. However, the above dosage does not limit the scope of the present invention in any way, since it can be increased or decreased depending on the route of administration, severity of obesity, gender, body weight, age, etc.

The compositions of the invention can be used alone or in combination with surgery, radiation therapy, hormonal therapy, chemotherapy, and methods using biological response modifiers.

Mesenchymal stem cell-derived exosomes (TSG-Exo) treated with endoplasmic reticulum stress inducers according to the present invention have increased levels of anti-inflammatory cytokines, regulatory T cells, and M2-type macrophages, and pro-inflammatory cytokines, T helper T cells. The immunomodulatory capacity was increased, including decreased levels of cells and M1-type macrophages, and effectively alleviated inflammation in a mouse model of colitis. Therefore, the present invention can be effectively used as a composition for preventing or treating inflammatory diseases and as a composition for immunomodulating.

Figure 1a shows the expression levels of MSC-specific antigens CD29, CD44, CD73, CD105, CD146 and CD34, CD45 by flow cytometry as cell surface markers and exosome (Exos) separation of WJ-MSCs. FIG. 1b shows a scheme for ultracentrifugation-based Exos separation as cell surface markers and exosome (Exos) separation of WJ-MSCs. Figure 2 shows the characteristics of naive (CTL-Exo) or TSG-primed WJ-MSC-derived exosomes (TSG-Exo). (A) Transmission electron microscopy (TEM) image of exosomes (scale bar = 100 nm); (B) Western blot analysis of CD63 and β-actin in WJ-MSCs and exosomes; (C) size distribution and concentration of exosomes by qNano; D) Exosome protein concentration by BCA protein analysis (*p<0.05, **p<0.01, ***p<0.001). FIG. 3 shows immunomodulatory factors in naive (CTL-Exo) or TSG-primed WJ-MSC-derived exosomes (TSG-Exo). (E) Relative mRNA expression levels of immunomodulatory factors by qRT-PCR; (F) Levels of TGFβ, IDO and COX2 by Western blot analysis; (G) CTL-Exo (+CTL-Exo) or TSG- by qRT-PCR. Relative mRNA expression levels of immunomodulatory factors in Con A-stimulated MNCs co-cultured with Exo (+TSG-Exo) for 48 hours; (H) Early apoptosis or late apoptosis of Con A-stimulated MNCs by flow cytometry. (*p<0.05, **p<0.01, ***p<0.001). FIG. 4 shows T cell proliferation regulation of TSG-primed WJ-MSC-derived exosomes (TSG-Exo) on activated peripheral blood mononuclear cells (PBMCs). (A) Representative histogram of proliferation of total T cells; (B) quantitative analysis for proliferation rate of total T, CD4+ T and CD8+ T cells; (C) fluorescence profile of CFSE-labeled cells in five individual PBMC sections. indicates division; Figure 5 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) regulate helper T cell (Th) differentiation and macrophage polarization. (D) Percentages of CD4+IFNγ+Th1, CD4+IL-17A+Th17 and CD4+CD25+Foxp3+Treg by flow cytometry; (E) Expression levels of M1 macrophage (CD80, CD86) or M2 macrophage (CD206, CD163) surface markers by flow cytometry; shown. Figure 6 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) regulate helper T cell (Th) differentiation, showing the percentage of Th1, Th, 17 and Treg cells by flow cytometry. . FIG. 7 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) regulate macrophage polarization, and shows representative images of M0, M1, and M2 (scale bar = 100 μm). Figure 8 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) modulate macrophage polarization and shows expression of M1 and M2 macrophage surface markers by flow cytometry. Figure 9 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) ameliorate DSS-induced colitis in mice. (A) Schematic image of the DSS-induced colitis experiment; (B) body weight; and (C) changes in disease activity index (DAI) scores; shown (n = 3 mice per group; *p<0.05 , **p<0.01, ***p<0.001). Figure 10 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) ameliorate DSS-induced colitis in mice. (D,E) Colon length measured by sacrificing mice on day 10; (F) H&E staining of colon sections (scale bar = 100 μm); (G) histological score; (H) colon MPO (n=3 mice per group; *p<0.05, **p<0.01, ***p<0.001). Figure 11 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) induce regulatory T cell and M2 macrophage polarization in vivo. (A) Th1, Th, 2, Th, 17 and Treg-related cytokines in colon tissue by qRT-PCR; (B) expression levels of macrophage phenotype-related genes; (*p<0.05, **p< 0.01, ***p<0.001). Figure 12 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) induce colonic lamina propria M2 macrophage polarization in vivo. Percentage of F4/80+CD11b+ macrophages in lamina propria cells of 3% DSS-colitis mouse colon analyzed (C) and quantified by flow cytometry; (E) F4/80+CD11b+ analyzed and quantified by flow cytometry. Expression of CD206 and Arginase 1 in macrophages; expressed as relative mean fluorescence intensity (RelMFI) normalized to control group (*p<0.05, **p<0.01, ***p<0.001) . Figure 13 shows the types of endoplasmic reticulum stress inducers (thapsigargin (TSG, Th, apsigargin), tunicamycin (Tu, Tunicamycin), brefeldin A (BFA), dithiothreitol (DTT) treated with human WJ-MSCs. , dithiothreitol) and MG132 (MG)). Treatment with endoplasmic reticulum stress inducers increased the expression levels of endoplasmic reticulum stress markers (GRP94) and anti-inflammatory cytokines (COX2, IDO, IL-10), but not pro-inflammatory cytokines (IL-1β, IFNγ, TNFα). We were able to confirm that the expression level of . FIG. 14 shows the results of confirming the suppression of T cell proliferation as an immunomodulatory effect of human WJ-MSCs treated with endoplasmic reticulum stress inducers (TSG, Tu, BFA, DTT, MG). It was confirmed that MSCs treated with an endoplasmic reticulum stress inducer had a further increased suppressive effect on the proliferation of total T, CD4+, and CD8+ T cells compared to MSCs that were not treated. FIG. 15 shows the results of confirming the suppression of T cell proliferation as an immunomodulatory effect of exosomes secreted from human WJ-MSCs treated with endoplasmic reticulum stress inducers (TSG, Tu, BFA, DTT, MG). It was confirmed that MSCs treated with TSG, Tu, or BFA among endoplasmic reticulum stress inducers had a further increased suppressive effect on the proliferation of total T, CD4+, and CD8+ T cells, compared to MSCs that were not treated.

[Example 1]
Confirmation of exosome characteristics derived from TSG-primed WJ-MSCs <1-1> WJ-MSC preparation and culture Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) were isolated and cultured based on Non-Patent Document 3. and sought to further characterize them based on surface marker expression profiles.

Specifically, with written consent from pregnant women and approval from the Institutional Review Board of Seoul Asan Hospital (protocol number 2015-3030), Wharton's jelly tissue was collected from the umbilical cord after normal term delivery. WJ-MSCs were isolated based on Non-Patent Document 1, and a mycoplasma test was performed. Cells were incubated at 37°C and 5% CO _in 10% FBS (Tissue Culture Biologicals, Tulare, CA), 1% Glutamax (Gibco), 25ng/ml EGF (Biolegend NS, Inc., San Diego, CA), 50ng /ml Cultured in DMEM (Gibco BRL, Grand Island, NY) with bFGF (Peprotech, Rocky Hill, NJ) and 1% antibiotic/antibacterial (Gibco). Upon reaching 80-90% confluence, cells were dissociated with 0.05% Trypsin-EDTA (Gibco) and further subcultured.

Flow cytometry results demonstrated that WJ-MSCs positively expressed MSC-specific cell surface markers CD29, CD44, CD73, CD105, and CD146, and negatively expressed hematopoietic stem cell-specific markers CD34 and CD45 (Figure 1a).

<1-2> WJ-MSC-derived exosome isolation and characterization Along with exosome-specific markers, pro-inflammatory and anti-inflammatory cytokine secretion patterns determine the immunomodulatory properties of MSCs or MSC-derived exosomes, thereby determining inflammatory genes. We attempted to confirm the expression of

WJ-MSC-derived exosomes (Exo(s)) were isolated from culture supernatants of naïve (CTL-Exo) or TSG-primed WJ-MSCs (TSG-Exo) by standard ultracentrifugation (Fig. 1b). . WJ-MSC cells (3×10 ⁶ ) were stimulated with 1 μM Thapsigargin (Thapsigargin, TSG; Sigma-Aldrich, St. Louis, MO) for 24 hours in 100 mm culture dishes. Thereafter, the medium was replaced with medium supplemented with 10% Exo-depleted FBS. Exo-depleted FBS was obtained by ultracentrifugation at 100,000xg for 18 hours. After 72 hours of culture, the culture fluid was collected and centrifuged at 300×g for 10 minutes, 2,500×g for 25 minutes, and 10,000×g for 1 hour at 4°C. The supernatant was filtered using a 0.22 μm filter and ultracentrifuged at 100,000×g for 2 hours. The Exo pellets were then washed with sterile PBS by centrifugation at 100,000×g for 2 hours, suspended in 200 μl of PBS, and stored at −80° C. until use. To suppress Exo production, cells were treated with culture medium containing 10 μM GW4869 (Santa cruz Biotechnology, Dallas, TX) for 12 hours. Thereafter, the medium was replaced with medium supplemented with 10% Exo-depleted FBS. After 72 hours, the culture supernatant was ultracentrifuged to separate Exo and stored at -80°C until use (concentrated-CM). Concentrated CM derived from GW4869-treated MSCs (Exo secretion suppressed) was used as a vehicle control group for all mouse experiments. The amount of Exo was determined with a BCA analysis kit (Thermo Fisher Scientific, Waltham, MA) for measurement of total protein. Exo size and number were determined with a qNano (Izon Science, Christchurch, New Zealand) and transmission electron microscopy (TEM; Jeol, Tokyo, Japan) based on the manufacturer's guidelines.

For Western blot analysis, WJ-MSC lysates and Exo were dissolved in RIPA buffer (Thermo Fisher) with protease inhibitors. Equal amounts of proteins were applied to SDS-PAGE and analyzed with primary antibodies for CD63 (SBI), COX2 (Abcam, Cambridge, UK), IDO (Merk Millipore, Darmstadt, Germany), TGFβ and GAPDH (Santa cruz). Bands were detected using enhanced chemiluminescence (Thermo Fisher) and images were captured using a LAS-3000 system (Fujifilm, Tokyo, Japan).

As a result, transmission electron microscopy analysis showed that purified exosomes contained round vesicles with a diameter of 50-120 nm (Figure 2A). The exosome-specific marker CD63 was expressed in CTL-Exo and TSG-Exo, but the cytoplasmic marker β-actin was not expressed in any exosomes (Figure 2B). As a result of qNano analysis, the average particle diameter of exosomes was 110 to 120 nm for both CTL-Exo and TSG-Exo (Figure 2C). The production of exosomes from TSG-primed WJ-MSCs was 50% even higher than that from naive WJ-MSCs (Fig. 2C,D). TSG-Exo had significantly higher expression levels of IL-10, TGFβ, COX2 and IDO, but lower expression of pro-inflammatory cytokines, including IFNγ, TNFα and IL-1β, compared to CTL-Exo ( Figure 3E). Consistent with the mRNA expression results, TGFβ, COX2 and IDO protein expression levels were significantly increased in TSG-Exo (Fig. 3F).

[Example 2]
Confirmation of the immunomodulatory effect of TSG-primed exosomes - suppression of T cell proliferation To evaluate the immunomodulatory potential of TSG-primed exosomes, we investigated anti-inflammatory cytokines and pro-inflammatory cytokines in activated mononuclear cells (MNCs). Cytokine expression levels were measured.

In quantitative RT-PCR, total RNA was extracted from cells, Exos, or tissue samples using TRizol reagent (Invitrogen, Waltham, MA), and cDNA was synthesized from total RNA using Superscript ^™ reverse transcriptase (Invitrogen). It was done. Quantitative PCR was measured using SYBR Green Master Mix (Applied Biosystems, Foster City, CA) in an MX300P thermocycler (Stratagene, San Diego, CA). The mRNA expression level was normalized to the level of GAPDH. Primer sequences for qRT-PCR are as listed in Table 1 below.

Mononuclear cells (MNCs) for apoptosis analysis were separated from hUCB using Ficoll-Hypaque (GE Healthcare, Chicago, IL) gradient centrifugation as described in [2] and supplemented with 10% Exo-depleted FBS. The cells were resuspended in RPMI 1640 medium. Umbilical cord blood (UCB) units were obtained from the Korea Catholic Hematopoietic Stem Cell Bank (CHSCB) from April 2019 to June 2020 with Institutional Review Board approval (IRB No. 2019-0467-0003). MNCs were stimulated with 5 μg/ml Concanavalin A and plated in 96-well plates (1× 10 ⁵ /well). Annexin V/7AAD (BD Biosciences, Franklin Lakes, NJ) staining was performed to determine cell apoptosis according to manufacturer's guidelines and analyzed by flow cytometry.

Next, to test the immunosuppressive effect of TSG-Exo on the proliferation of activated peripheral blood-derived mononuclear cells (PBMCs), human peripheral blood from healthy donors was collected from the Korean Red Cross Society (Seoul, Korea). Provided with the approval of the Institutional Review Board of Catholic University of America (IRB No. 2019-2891-0003). PBMC were isolated by centrifugation through a Ficoll-Hypaque density gradient and resuspended in RPMI 1640 medium supplemented with 10% Exo-depleted FBS.

T cell proliferation was quantified by CFSE dilution. The separated PBMCs were labeled with 2 μM CFSE (CellTrace CFSE Cell Proliferation Kit, Th, ermo Fisher Scientific). CFSE-labeled PBMC (1×10 ⁵ /well) were naive or TSG-primed in the presence of anti-CD3/CD28 microbeads (Gibco) and recombinant human IL-2 (30 U/ml, Peprotech). The cells were co-cultured in 96-well plates with or without Exos (4 μg) derived from WJ-MSCs. After culturing for 6 days, the cells were stained with human monoclonal antibodies fluorescently labeled to CD45, CD3, CD4, or CD8 (BD Biosciences), and measured by flow cytometry. All cells were gated on 7AAD negative cells.

As a result, treatment of MNCs stimulated with Con A (concanavalin A) with exosomes increased the expression levels of COX2, NOS2, IDO, IL-10, and TGFβ, whereas the expression levels of TNFα and IL-1β decreased. Diminished. This was more pronounced in activated MNCs treated with TSG-Exo than in those treated with CTL-Exo (Fig. 3G). TSG-Exo slightly reduced early apoptosis of MNCs, but had no effect on late apoptosis. This suggests that exosome treatment does not induce cytotoxicity (Figure 3H). CFSE dilution analysis showed that the addition of TSG-Exo inhibited the proliferation of total T, CD4+ T and CD8+ T cells more strongly than CTL-Exo (Fig. 4A,B). In TSG-Exo-treated PBMCs, the proportion of cells in early division cycle 1 was significantly increased, and the proportion of cells in division cycles 4 and 5 was significantly decreased (Figure 4C). These results suggest that TSG-primed exosomes increase the production of anti-inflammatory cytokines and have a significant suppressive effect on T cell proliferation.

[Example 3]
Confirmation of immunomodulatory effects of TSG-primed exosomes - Improvement of regulatory T cell and M2 type macrophage polarization <3-1> Confirmation of T helper cell (Th) differentiation of TSG-primed exosomes MSC exosomes are It has been reported that immunomodulatory effects can be mediated through differentiation of cells (Tregs). Therefore, we investigated T helper cell (Th) differentiation to confirm the effects of TSG-Exo on specific T cell subsets.

Th cells were induced by stimulation with family-specific cytokines on CD4+ cells purified from human PBMC in the presence of exosomes. Specifically, isolation of CD4+ T cells from PBMC lymphocytes was performed with a human CD4 T cell isolation kit (Miltenyi Biotec, Marburg, Germany) based on the manufacturer's protocol. CD4+ T cells were stimulated with anti-CD3/CD28 microbeads and IL-2 (20 ng/ml), and for specific Th cell differentiation, specific cytokines were added: IFNγ ml for Th1) and IL-12 (25 ng/ml). ); IL-6 (50 g/ml) and TGFβg/ml for Th17; TGFβg/ml for Tregs) and retinoic acid (10 nM). Cytokine-treated CD4+ T cells (5×10 ⁵ /well) were cultured for 5 days in a 24-well plate with the addition of naive or TSG-primed WJ-MSC-derived Exos. For intracellular staining, cells were stimulated with cell stimulation cocktail (Invitrogen) for 5 hours before staining. Cells were stained with cell surface markers anti-CD3, anti-CD4, and anti-CD25 antibodies, then fixed/permeabilized and stained with fluorescently labeled anti-IFNγ, anti-IL-17A, and anti-Foxp3 antibodies (BD Biosciences). Stained cells were analyzed by flow cytometry.

As a result, co-culture with TSG-Exo greatly reduced the induction of IFNγ+CD4+Th1 and IL-17A+CD4+Th17 cells and increased Foxp3+CD25+CD4+Treg cells compared to CTL-Exo (Figure 5D and Figure 6). M1-type macrophages exhibited a round morphology, whereas M2-type macrophages exhibited a spindle-like morphology (FIG. 7).

<3-2> Confirmation of enhancement of M2-type macrophage polarization by TSG-primed exosomes It was reported that MSC exosomes can mediate immunomodulatory effects through induction of M2-type macrophage polarization. Therefore, we investigated whether TSG-Exo could modulate the macrophage phenotype to confirm the effect of TSG-Exo on specific T cell subsets.

GM-CSF (for M1 type) or M-CSF (for M2 type) stimulated primary human macrophages were cultured with exosomes in the presence of M1 type cytokines or M2 type cytokines. Specifically, CD14+ mononuclear cells were separated from hUCB-derived MNCs using CD14-conjugated microbeads (Miltenyi Biotec). To generate macrophages, mononuclear cells (5 × 10 ⁵ /well) were incubated with GM-CSF (50 ng/ml; Peprotech) or M-CSF (100 ng/ml) in RPMI 1640 medium containing 10% FBS (Gibco ) for 6 days. For M1 polarization in 24-well plates, GM-CSF-derived macrophages were stimulated with IFNγng/ml)+LPS (1 μg/ml) for 48 hours with naive or TSG-primed WJ-MSC-derived Exo. Ta. For M2 polarization, M-CSF-derived macrophages were stimulated with IL-4 (20 ng/ml) and IL-13 (20 ng/ml) along with Exo from naive or TSG-primed WJ-MSCs for 48 h. It was done. After co-culture, cells were stained with fluorescently labeled human monoclonal antibodies for CD14, CD80, CD86, CD206 and CD163 (BD Biosciences) and analyzed by flow cytometry.

As a result, M1 type cell surface markers CD80+ and CD86+ were significantly decreased in M1 type macrophages cultured with TSG-Exo, whereas those cultured with CTL-Exo. However, the expression of M2-type cell surface markers CD206+ and CD163+ increased when cultured with exosomes, and this effect was even greater in the TSG-Exo treated group (Fig. 5E and Fig. 8). Such results indicate that TSG-Ex
This suggests that o induces M2-type macrophage phenotype more effectively than naive exosomes. Therefore, TSG treatment on MSCs can generate exosomes with improved immunomodulatory properties.

[Example 4]
Confirmation of the DSS-induced colitis alleviation effect of TSG-primed exosomes We confirmed the potential protective effect of TSG-Exo in a 3% DSS-induced colitis mouse model (Fig. 9A ).

Specifically, for the production of a DSS-induced colitis mouse model, 3% (w/v) DSS (Dextran sulfate) was added to the drinking water of 7-week-old C57BL/6 mice for 7 days.
induction by administering sodium; MP Biomedicals, Santa Ana, CA). The mice were divided into 4 groups of 10 mice each: (1) control group (negative control group), (2) DSS (positive control group) administered with concentrated CM extracted from GW4869-treated MSCs (solvent control group, 200 μl). control group), (3) DSS administered with Exo derived from naive MSCs, (4) DSS administered with Exos derived from TSG-primed MSCs. For groups (3) and (4), mice were injected intraperitoneally with 200 μg of Exo diluted in 200 μl PBS on days 1, 3, and 5. Mice were euthanized on day 10. Weight loss (0-4), stool density (0-4), stool blood (0-4), coat roughness (0-4), rectal prolapse (0-3), bent posture (0 -3), the severity of colitis using the disease activity index (DAI), which includes bedding contamination (0-2) and not inquisitive/alert (0-2). The level was evaluated daily. All animal experimental procedures were approved by the Animal Research Ethics Committee of Catholic University of America (IACUC no. 2019-0301-03). As a vehicle control group, MSC culture supernatant (GW-CM) treated with GW4869 (10 μM), which is an nSMase 2 (neutral sphingomyelinase supernatant 2) inhibitor that regulates exosome secretion, was used.

The colon length of mice sacrificed on day 10 was measured. Colon tissue was fixed in 4% formaldehyde (Wako, Osaka, Japan), embedded in paraffin, and then sectioned at 5 μm. For histological analysis, sections of the colon were stained with hematoxylin and eosin (H&E). Histopathology scores were determined in a blinded manner according to the degree of inflammatory cell infiltration (0-4) and the degree of tissue damage (0-4).

The infiltration of neutrophils into the colon tissue was measured using myeloperoxidase (MPO) Colorimetric Activity Assay Kit (Sigma). The colon was homogenized with MPO analysis buffer and the supernatant was collected by centrifugation at 13,000xg for 10 min. The supernatant was mixed with MPO assay buffer and MPO substrate, incubated at 25°C for 120 minutes, and then TNB (tetramethylbenzidine) probe was added. Absorbance was measured at 412 nm using a spectrophotometer (Biotek, Winooski, WT).

As a result, GW4869 successfully suppressed exosome release in MSCs. GW-CM treated colitis mice showed sustained weight loss and significantly elevated disease activity index (DAI) such as stool consistency, bloody diarrhea and general activity. However, exosome treatment improved weight loss and DAI especially in the TSG-Exo treated group (Fig. 9B,C). Furthermore, the shortening of colon length by DSS administration was significantly improved in TSG-Exo-treated mice compared to CTL-Exo-treated mice (Fig. 10D,E). Histological examination showed that TSG-Exo-treated mice maintained colonic tissue integrity; significant structural disruption, loss of crypts and reduced infiltration of inflammatory cells; and CTL-Exo had even lower histological scores compared to treated mice (Fig. 10F,G). Furthermore, MPO (myeloperoxidase) activity, which indicates neutrophil infiltration in mice sacrificed on day 10, was significantly decreased in TSG-Exo-treated mice compared to CTL-Exo-treated mice (Fig. 10H). These results indicate that TSG-Exo had improved protective activity against intestinal inflammation in DSS-induced colitis compared to naive exosomes.

[Example 5]
Confirmation of the effect of TSG-primed exosomes on enhancing regulatory T cell and M2 macrophage polarization in the inflamed colon Immune cell responses play an important role in the pathogenesis of inflammatory bowel disease (IBD). In particular, the balance between Tregs and other T cells in the intestinal microenvironment plays an important role in alleviating colitis, and macrophages mediate the inflammatory response through M1/M2 polarization in the colon, leading to the pathogenesis of IBD. plays an important role in the mechanism. Therefore, we sought to confirm the immunomodulatory effect of TSG-primed exosomes on the polarization of regulatory T cells and M2-type macrophages in the inflamed colon. We also next investigated the effect of TSG-primed exosomes on the immune cell profile of mice with DSS-induced colitis.

Specifically, 3% DSS-colitis mice (n = 2-4 mice per group) were sacrificed on day 10 and colon tissues were harvested. and Treg cell levels were assessed. Lamina propria cells were isolated from the intestine. Colon tissues were cut into small pieces (3 mm x 3 mm) and incubated with HBSS buffer containing 2 mM EDTA (Sigma) for 30 min at 220 rpm in a shaking incubator. After removing the epithelium at 37°C, tissues were cultured in RPMI containing 5% FBS, 1 mg/ml Collagenase D (Sigma) and 0.1 mg/ml DNase I (Sigma) with shaking at 220 rpm for 60 min at 37°C. It was digested in a container. The degraded samples were filtered through a 40 μm cell strainer and then centrifuged at 300×g for 5 minutes. The separated cells were stained with fluorescently labeled antibodies against CD11b, F4/80, CD206, and arginase 1 (BD Biosciences) and analyzed by flow cytometry.

As a result, the expression levels of Th1 (T-bet), Th2 (GATA3) and Th17 (RORγ family transcription factors) were increased in the colon tissue of GW-CM treated mice, but decreased in exosome treated mice. In particular, GATA3 expression was further significantly decreased in TSG-Exo-treated mice compared to CTL-Exo-treated mice.Foxp3 and Treg transcription factors were significantly reduced in TSG-Exo-treated mice compared to CTL-Exo-treated mice. (Fig. 11A).

Also, M1 type marker expression of CXCL9, MCP1 and iNOS was decreased in CTL-Exo- and TSG-Exo treated mice compared to GW-CM treated mice. CXCL9 levels were significantly decreased in TSG-Exo treated mice compared to CTL-Exo treated mice. M2 type marker expression of Arg1 and CD206 was increased upon exosome treatment, and this effect was significantly greater in the TSG-Exo treated group (FIG. 11B).

It was found that mice with DSS-induced colitis had a significantly increased proportion of F4/80+CD11b+ cells (macrophages) in the lamina propria of the large intestine compared to mice in the negative control group (FIGS. 12C, D). Although exosome treatment did not affect the percentage of F4/80+CD11b+ cells (Fig. 12C,D), the expression levels of CD206 and Arginase 1 in colonic macrophages were significantly increased in TSG-primed exosome-treated mice (Fig. 12E). These data suggest that TSG-primed exosomes attenuate mucosal inflammation by inducing regulatory T cells and polarizing M2-type macrophages.

[Example 6]
ER stress-induced WJ-MSC or exosome analysis <6-1> Confirmation of ER stress-induced WJ-MSC characteristics In order to investigate the influence of endoplasmic reticulum stress (ER-stress) on the immunomodulatory properties of human MSC, we - Thapsigargin (TSG, Th, apsigargin), Tunicamycin (Tu), Brefeldin A (BFA), dithiothreitol (DTT) or MG132 (MG) was used as a stress inducer in the above Example <1- 2>, the expression of ER stress markers and inflammatory genes was confirmed in WJ-MSCs in which ER stress was induced.

As a result, as shown in FIG. 13, ER-stress markers GRP94 and CHOP significantly increased after treatment with ER-stress inducers in human MSCs. Additionally, MSCs treated with ER-stress inducers had significantly higher expression levels of anti-inflammatory cytokines COX2, IDO, and IL-10 than untreated MSCs, while pro-inflammatory cytokines including IL-1β, IFNγ, and TNFα We confirmed that the expression of cytokines was even lower.

<6-2> Confirmation of the immunomodulatory effect of ER stress-induced WJ-MSCs - Suppression of T cell proliferation. For evaluation, PBMC and MSC were co-cultured in the same manner as in Example <1-2> above.

As a result, as shown in Figure 14, MSCs treated with ER-stress inducers (TSG, Tu, BFA, DTT, MG) had higher total T, CD4+, and CD8+ T cells than MSCs that were not treated. It was confirmed that the growth inhibitory effect was further increased.

These results indicate that MSCs subjected to ER stress by an ER-stress inducer increase the production of anti-inflammatory cytokines and exhibit an effective function in enhancing the suppression of T cell proliferation.

<6-3> Confirmation of the immunomodulatory effect of exosomes secreted from ER stress-induced WJ-MSCs - Suppression of T cell proliferation from MSCs treated with ER stress inducers (TSG, Tu, BFA, DTT, MG) In order to confirm the immunosuppressive effect of secreted exosomes, PBMC and exosomes were co-cultured in the same manner as in [Example 2] above.

As a result, as shown in Figure 15, exosomes treated with TSG, BFA, and Tu more strongly suppressed the proliferation of total T, CD4+T, and CD8+T cells compared to the control group (cont-exo). I was able to confirm that.

These results indicate that ER-stress inducers can generate exosomes with improved immunomodulatory properties in MSCs.

[Statistical analysis]
Statistical analyzes were performed using GraphPad Prism v8.0.1 (GraphPad Software, San Diego, CA) using member analysis of variance (ANOVA) and Tukey's multiple comparisons test. All data are mean±S. Displayed as D. P values <0.05 were considered statistically significant.

Mesenchymal stem cell-derived exosomes (TSG-Exo) treated with endoplasmic reticulum stress inducers according to the present invention increase the levels of anti-inflammatory cytokines, regulatory T cells, and M2-type macrophages, and increase the levels of pro-inflammatory cytokines and helper cells. The immunomodulatory capacity was increased, including decreased levels of T cells and M1-type macrophages, and effectively alleviated inflammation in a mouse model of colitis. Therefore, the present invention has industrial applicability because it can be effectively used as a composition for preventing or treating inflammatory diseases and as a composition for immunomodulating.

Sequence catalog Free Text
SEQ ID NO: 1 corresponds to the forward primer sequence of hCOX-2.
SEQ ID NO: 2 corresponds to the reverse primer sequence of hCOX-2.
SEQ ID NO: 3 corresponds to the hIDO forward primer sequence.
SEQ ID NO: 4 corresponds to the hIDO reverse primer sequence.
SEQ ID NO: 5 corresponds to the forward primer sequence of hIFNγ.
SEQ ID NO: 6 corresponds to the reverse primer sequence of hIFNγ.
SEQ ID NO: 7 corresponds to the forward primer sequence of hIL-10.
SEQ ID NO: 8 corresponds to the reverse primer sequence of hIL-10.
SEQ ID NO: 9 corresponds to the forward primer sequence of hIL-1β.
SEQ ID NO: 10 corresponds to the reverse primer sequence of hIL-1β.
SEQ ID NO: 11 corresponds to the forward primer sequence of hNOS2.
SEQ ID NO: 12 corresponds to the reverse primer sequence of hNOS2.
SEQ ID NO: 13 corresponds to the forward primer sequence of hTGFβ.
SEQ ID NO: 14 corresponds to the reverse primer sequence of hTGFβ.
SEQ ID NO: 15 corresponds to the forward primer sequence of hTNFα.
SEQ ID NO: 16 corresponds to the reverse primer sequence of hTNFα.
SEQ ID NO: 17 corresponds to the forward primer sequence of mArg1.
SEQ ID NO: 18 corresponds to the reverse primer sequence of mArg1.
SEQ ID NO: 19 corresponds to the forward primer sequence of mCD206.
SEQ ID NO: 20 corresponds to the reverse primer sequence of mCD206.
SEQ ID NO: 21 corresponds to the forward primer sequence of mCXCL9.
SEQ ID NO: 22 corresponds to the reverse primer sequence of mCXCL9.
SEQ ID NO: 23 corresponds to the forward primer sequence of mFoxp3.
SEQ ID NO: 24 corresponds to the reverse primer sequence of mFoxp3.
SEQ ID NO: 25 corresponds to the forward primer sequence of mGATA3.
SEQ ID NO: 26 corresponds to the reverse primer sequence of mGATA3.
SEQ ID NO: 27 corresponds to the forward primer sequence of miNOS.
SEQ ID NO: 28 corresponds to the miNOS reverse primer sequence.
SEQ ID NO: 29 corresponds to the forward primer sequence of mMCP1.
SEQ ID NO: 30 corresponds to the reverse primer sequence of mMCP1.
SEQ ID NO: 31 corresponds to the forward primer sequence of mRORγ.
SEQ ID NO: 32 corresponds to the reverse primer sequence of mRORγ.
SEQ ID NO: 33 corresponds to the forward primer sequence of mT-bet.
SEQ ID NO: 34 corresponds to the reverse primer sequence of mT-bet.

Claims (10)

A pharmaceutical composition for preventing or treating inflammatory diseases, comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress-inducing substance. The endoplasmic reticulum stress inducer is one or more selected from the group consisting of thapsigargin (TSG, Th, apsigargin), tunicamycin, brefeldin A, dithiothreitol (DTT), and MG132. The pharmaceutical composition according to claim 1, characterized in that: The mesenchymal stem cells may be any cell selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tubes, corneal stroma, lungs, muscle, and fetal liver. 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a mesenchymal stem cell obtained from one or more tissues. The pharmaceutical composition according to claim 1, wherein the size of the exosome is 10 to 200 nm. The inflammatory disease is any one or more selected from the group consisting of acute or chronic inflammatory disease, chronic bronchitis, rhinitis, arthritis, autoimmune disease, transplant rejection, and inflammatory bowel disease. Item 1. Pharmaceutical composition according to item 1. An immunomodulatory composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer. The immunomodulation increases the levels of anti-inflammatory cytokines, regulatory T cells and M2-type macrophages, and pro-inflammatory cytokines. ), helper T cells and M1-type macrophage. A method for promoting the production of exosomes derived from mesenchymal stem cells, which includes the step of treating mesenchymal stem cells with an endoplasmic reticulum stress-inducing substance. A method for treating an inflammatory disease, comprising administering to a patient with an inflammatory disease a composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer. Use of a composition comprising mesenchymal stem cell-derived exosomes treated with an endoplasmic reticulum stress inducer for use in the prevention or treatment of inflammatory diseases.