Exosomes Derived from Cells Treated with Endoplasmic Reticulum Stress Inducers and Their Use

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases, containing exosomes derived from mesenchymal stem cells induced with endoplasmic reticulum stress, and its use. Specifically, the immunomodulatory factors in the exosomes derived from WJ-MSCs of the present invention are increased by treatment with endoplasmic reticulum stress inducers 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 a murine colitis model. Furthermore, administration of TSG-primed WJ-MSC-derived exosomes improved inflammatory responses 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

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2020-0174576, filed on December 14, 2020, and the entire specification is incorporated herein by reference.

[0002] The present invention relates to the therapeutic use of exosomes derived from endoplasmic reticulum stress-induced mesenchymal stem cells.

Background Art

[0003] Mesenchymal stem cells (MSCs) are known to have immunomodulatory properties, which are mediated by immunosuppression, vascular regulation, and the activity of paracrine factors. Known paracrine secretomes include the secretion of immunomodulatory cytokines, growth factors, and small membrane vesicles. However, the direct use of mesenchymal stem cells has problems such as maintaining their phenotype and functional stability, or high separation and handling costs.

[0004] Among the mesenchymal stem cell secretomes, exosomes mediate the transmission of paracrine factors in cell communication and play an important role in immunomodulation. Exosomes are small membrane lipid vesicles with a size of 30-150 nm released into the extracellular space, which transmit proteins, mRNA, microRNA (miRNA), or other components to target cells and promote the activation of immunomodulatory pathways. In particular, exosomes secreted from mesenchymal stem cells are known to exhibit the regenerative medical therapeutic efficacy of stem cells. The functions and transmitted elements of exosomes vary depending on cell type, and cellular stress can affect the elements transmitted by exosomes.

[0005] The endoplasmic reticulum plays an important role in regulating signal transduction pathways to ensure cellular homeostasis. Newly synthesized proteins undergo folding and translocation through the ER membrane. However, when the ER fails to maintain homeostasis under stressful pathological and physiological conditions, the unfolded protein response (UPR) is activated, which can affect cellular functions. Prolonged ER dysfunction or stress is involved in the etiology of various diseases, including metabolic and inflammation-related diseases. Thapsigargin (TSG) is known to increase cytoplasmic calcium concentration and deplete ER calcium stores, thereby inducing ER stress.

[0006] Korean Registered Patent No. 10-1980453 relates to a composition for promoting the production of exosomes derived from stem cells, and discloses a composition and method for increasing the production amount of exosomes derived from stem cells and the content of proteins and RNAs in exosomes using pioglitazone, metformin, or AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide). However, there is no disclosure of research or description regarding exosomes secreted from stem cells treated with ER stress inducers such as thapsigargin.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0008] As a result of intensive efforts to provide the secretome of mesenchymal stem cells as an alternative to treatments using mesenchymal stem cells directly, the inventors of the present invention have confirmed that in the case of exosomes secreted from mesenchymal stem cells induced to have endoplasmic reticulum stress with an endoplasmic reticulum stress inducer such as thapsigargin, they are involved in the activation and polarization of T cells and macrophages, and thus completed the present invention.

[0009] Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating an inflammatory disease, which contains exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer, and uses thereof.

Means for Solving the Problems

[0010] The present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, which contains exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

[0011] According to a preferred embodiment of the present invention, the endoplasmic reticulum stress inducer may be any one or more selected from the group consisting of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT), and MG132.

[0012] According to a preferred embodiment of the present invention, the mesenchymal stem cells may be mesenchymal stem cells obtained from any one or more tissues selected from the group consisting of adipose tissue, bone marrow, cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, and fetal liver.

[0013] According to a preferred embodiment of the present invention, the size of the exosomes may be 10 to 200 nm.

[0014] According to a preferred embodiment of the present invention, the inflammatory disease is selected from the group consisting of acute or chronic inflammatory diseases, chronic bronchitis, rhinitis, arthritis, autoimmune diseases, transplant rejection, and inflammatory bowel diseases. Any one or more thereof may be selected.

[0015] The present invention also provides a composition for immunomodulation comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

[0016] According to a preferred embodiment of the present invention, the immunomodulation may be an immunomodulation that increases the levels of anti-inflammatory cytokines, regulatory T cells, and M2-type macrophages and decreases the levels of pro-inflammatory cytokines, helper T cells, and M1-type macrophages. cytokine), helper T cell and M1-type macrophage levels.

[0017] The present invention also provides 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 inducer.

[0018] The present invention also provides a method for treating an inflammatory disease, which includes the step of administering to a patient with an inflammatory disease a composition containing exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

[0019] The present invention also provides the use of a composition containing exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer for use in the prevention or treatment of inflammatory diseases.

[0020] Exosomes derived from mesenchymal stem cells (MSCs) have low immunogenicity compared to parental MSCs because of their low content of membrane-bound proteins including tetraspanins (CD81, CD63, CD9), heat shock proteins (HSP60, HSP70, HSP90), and TSG101, and are known not to cause rejection by activating allogeneic immune responses in MHC-mismatched recipients.

[0021] Therefore, in order to apply MSC-derived exosomes to the treatment of immune-mediated inflammatory diseases, the present inventors confirmed that exosomes (TSG-Exo) secreted from human Wharton's jelly-derived MSCs (WJ-MSCs) treated with thapsigargin (TSG), an endoplasmic reticulum (ER) stress inducer, have improved immunomodulatory properties.

[0022] When WJ-MSCs were stimulated with TSG, exosome secretion increased, and the yields and expressions of immunomodulatory and anti-inflammatory factors such as IL-10, COX2, or IDO (Indoleamine 2,3-dioxygenase) increased, while the expression levels of pro-inflammatory factors (IFNγ, TNFα, or IL-1β) decreased or were maintained similarly. Similar results were confirmed with TSG-Exo, suggesting that TSG-induced exosome release has a secretion mechanism different from the inflammation- or aging-related secretory mechanism (SASP).

[0023] In addition, even though TSG-Exo is derived from human MSCs, no clear immune rejection reaction was observed when administered to a mouse colitis model, meaning that TSG-Exo has immune tolerance by the host even in a heterologous system, which is advantageous for future clinical application. TSG-Exo substantially alleviated colitis by reducing the inflammatory response and maintaining intestinal barrier integrity in a mouse colitis model. A significant increase in Tregs and M2-type macrophages in the colon of colitis mice treated with TSG-Exo suggests the anti-inflammatory effect of TSG-Exo. Also, Con When treated with A-stimulated MNC (mononuclear cells), the anti-inflammatory factors of exosomes were efficiently transmitted to MNC, so similar expressions occurred in exosomes and exosome-treated MNC. Since TSG-Exo showed better regulation of T cell / macrophage polarization than control group exosomes, the secretome of exosomes can be a reliable indicator for predicting the therapeutic effect of exosomes.

[0024] For TSG-Exo, the proliferation of T cells derived from human peripheral blood and the inhibitory effects on Th1 and Th17 differentiation were significantly improved, while regulatory T cells and M2 macrophages were more abundant compared to the control group exosome-treated group. This suggests that intestinal Tregs and macrophages can be mediators for the anti-inflammatory effect of MSC exosomes. The upward regulation of Tregs and M2 macrophages maintained intestinal homeostasis as proposed by the disease activity score, histological score, and neutrophil activity reduction.

[0025] Such data means that TSG treatment is sufficient to induce the release of exosomes from WJ-MSCs and promotes the immunomodulatory properties of TSG-Exo, indicating that TSG-treated MSCs or TSG-Exo can provide a new treatment method for the treatment of colitis.

[0026] Therefore, the present invention can provide a pharmaceutical composition for preventing or treating inflammatory diseases containing exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

[0027] According to a preferred embodiment of the present invention, the endoplasmic reticulum stress inducer may be any one or more selected from the group consisting of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT), and MG132.

[0028] According to a preferred embodiment of the present invention, the mesenchymal stem cells may be mesenchymal stem cells obtained from any one or more tissues selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, and fetal liver. Preferably, the mesenchymal stem cells may be mesenchymal stem cells obtained from Wharton's jelly.

[0029] According to a preferred embodiment of the present invention, the size of the exosomes may be 10 to 200 nm. Preferably, the size of the exosomes may be 30 to 150 nm, and more preferably, 50 to 120 nm.

[0030] According to a preferred embodiment of the present invention, the inflammatory disease may be any one or more selected from the group consisting of acute or chronic inflammatory diseases, chronic bronchitis, rhinitis, arthritis, autoimmune diseases, transplant rejection, and inflammatory bowel diseases. Preferably, the inflammatory disease may be inflammatory bowel disease, and the inflammatory bowel disease may be ulcerative colitis, Crohn's disease, or Behçet's disease.

[0031] The present invention also provides an immunomodulatory composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

[0032] According to a preferred embodiment of the present invention, the immunomodulation increases the levels of anti-inflammatory cytokines, regulatory T cells, and M2-type macrophages, and reduces the levels of pro-inflammatory cytokines, helper T cells, and M1-type macrophages.

[0033] The present invention can also provide a method for promoting the production of exosomes derived from mesenchymal stem cells, which includes a step of treating the mesenchymal stem cells with an endoplasmic reticulum stress inducer.

[0034] The said promotion of production may mean promoting the number of exosomes and the production of exosome proteins and mRNAs.

[0035] The present invention can also provide a method for treating an inflammatory disease, which includes a step of administering a composition containing exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer to a patient with the inflammatory disease.

[0036] According to a preferred embodiment of the present invention, the endoplasmic reticulum stress inducer may be any one or more selected from the group consisting of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT), and MG132.

[0037] According to a preferred embodiment of the present invention, the mesenchymal stem cells may be mesenchymal stem cells obtained from any one or more tissues selected from the group consisting of adipose tissue, bone marrow, cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, and fetal liver. Preferably, the mesenchymal stem cells may be mesenchymal stem cells obtained from Wharton's jelly.

[0038] According to a preferred embodiment of the present invention, the size of the exosomes may be 10 - 200 nm. Preferably, the size of the exosomes may be 30 - 150 nm, and more preferably, 50 - 120 nm.

[0039] According to a preferred embodiment of the present invention, the inflammatory disease may be any one or more selected from the group consisting of acute or chronic inflammatory diseases, chronic bronchitis, rhinitis, arthritis, autoimmune diseases, transplant rejection, and inflammatory bowel diseases. Preferably, the inflammatory disease may be an inflammatory bowel disease, and the inflammatory bowel disease may be ulcerative colitis, Crohn's disease, or Behçet's disease.

[0040] The present invention can also provide the use of exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer for preventing or treating inflammatory diseases in a composition.

[0041] According to a preferred embodiment of the present invention, the endoplasmic reticulum stress inducer may be any one or more selected from the group consisting of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT), and MG132.

[0042] According to a preferred embodiment of the present invention, the mesenchymal stem cells may be mesenchymal stem cells obtained from any one or more tissues selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, and fetal liver. Preferably, the mesenchymal stem cells may be mesenchymal stem cells obtained from Wharton's jelly. It may also be mesenchymal stem cells obtained from jelly.

[0043] According to a preferred embodiment of the present invention, the size of the exosomes may be 10 - 200 nm. Preferably, the size of the exosomes may be 30 - 150 nm, and more preferably, 50 - 120 nm.

[0044] According to a preferred embodiment of the present invention, the inflammatory disease may be any one or more selected from the group consisting of acute or chronic inflammatory diseases, chronic bronchitis, rhinitis, arthritis, autoimmune diseases, transplant rejection, and inflammatory bowel diseases. Preferably, the inflammatory disease may be an inflammatory bowel disease, and the inflammatory bowel disease may be ulcerative colitis, Crohn's disease, or Behçet's disease.

[0045] Hereinafter, the definitions of the terms used in the present specification are as follows.

[0046] "Prevention" of the present invention means all actions that suppress an inflammatory disease or delay its onset.

[0047] "Improvement" or "treatment" of the present invention means all actions that make parameters related to an inflammatory disease, such as the degree of symptoms, improve or become beneficial by exosomes derived from mesenchymal stem cells treated with the endoplasmic reticulum stress inducer of the present invention.

[0048] The pharmaceutical composition of the present invention may be in various 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, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrants or surfactants, diluents or excipients can be used for preparation.

[0049] Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspension solvents, emulsions, lyophilized preparations, or suppositories, etc. As non-aqueous solvents and suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As the base of suppositories, witepsol, macrogol, tween 61, cocoa butter, laurin fat, glycerol, gelatin, etc. can be used.

[0050] The composition of the present invention can be administered parenterally and formulated by methods known in the art in the form of an injection for intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection during parenteral administration.

[0051] In the case of the injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. In the case of an injection, examples of suitable carriers include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or vegetable oils. More preferably, as a suitable carrier, Hank's solution, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% dextrose can be used. In order to protect the injection from contamination by microorganisms, it may further contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Also, the injection may often further contain an isotonic agent such as sugar or sodium chloride.

[0052] The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined by factors including the type and severity of the patient's disease, the activity of the drug, drug sensitivity, administration time, administration route and excretion ratio, treatment duration, elements including co-administered drugs, and other elements well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single or multiple doses. That is, the total effective amount of the composition of the present invention can be administered to a patient in a single dose or by a fractionated treatment protocol with multiple doses administered over a long period. Considering all the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0053] The dosage of the pharmaceutical composition of the present invention varies within a wide range depending on the patient's body weight, age, gender, health status, diet, administration time, administration method, excretion rate, and severity of the disease. As the daily dosage, when administered parenterally, based on exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per kg of body weight per day. Also, when administered orally, based on exosomes derived from mesenchymal stem cells treated with the endoplasmic reticulum stress inducer of the present invention, it is preferably administered in an amount of 0.01 to 100 mg, more preferably 0.01 to 10 mg per kg of body weight per day, and can be administered in 1 to several divided doses. However, since it can be increased or decreased according to the administration route, severity of obesity, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0054] The composition of the present invention can be used alone or in combination with methods using surgery, radiotherapy, hormone therapy, chemotherapy, and biological response modifiers.

Advantages of the Invention

[0055] Exosomes (TSG-Exo) derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer according to the present invention increased the levels of anti-inflammatory cytokines, regulatory T cells, and M2 macrophages, and decreased the levels of pro-inflammatory cytokines, helper T cells, and M1 macrophages, etc., increasing the immunomodulatory ability and effectively alleviating inflammation in a colitis mouse model. Therefore, the present invention can be effectively used as a composition for preventing or treating inflammatory diseases and a composition for immunomodulation.

Brief Description of the Drawings

[0056]

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Best Mode for Carrying Out the Invention

[0057] [Example 1] Confirmation of Characteristics of Exosomes Derived from TSG-Primed WJ-MSCs <1-1> Preparation and Culture of WJ-MSCs Mesenchymal stem cells (WJ-MSCs) derived from Wharton's jelly were isolated and cultured based on Non-Patent Document 3 and further characterized based on the surface marker expression profile.

[0058] Specifically, with the written consent of pregnant women and approval from the Institutional Review Board of Seoul Asan Hospital (protocol number 2015-3030), after normal full-term delivery, the tissue of Wharton's jelly was obtained from the umbilical cord. WJ-MSCs were isolated based on Non-Patent Document 1 and mycoplasma tests were performed. The cells were cultured in DMEM (Gibco BRL, Grand Island, NY) with 10% FBS (Tissue Culture Biologicals, Tulare, CA), 1% Glutamax (Gibco), 25 ng / ml EGF (Biolegend NS, Inc., San Diego, CA), 50 ng / ml bFGF (Peprotech, Rocky Hill, NJ), and 1% antibiotic / antimycotic (Gibco) at 37 °C and 5% CO2. When 80-90% confluence was reached, the cells were separated with 0.05% Trypsin-EDTA (Gibco) and further subcultured.

[0059] 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).

[0060] <1-2>Isolation and Characterization of Exosomes Derived from WJ-MSCs Together with exosome-specific markers, the immune regulatory properties of MSCs or MSC-derived exosomes were determined by the pro-inflammatory and anti-inflammatory cytokine secretion patterns to confirm the expression of inflammatory genes.

[0061] WJ-MSC-derived exosomes (Exo(s)) were isolated from the culture supernatants of naive (CTL-Exo) or TSG-primed WJ-MSCs (TSG-Exo) by standard ultracentrifugation (Figure 1b). WJ-MSC cells (3×10 6) was stimulated with 1 μM thapsigargin (TSG; Sigma-Aldrich, St. Louis, MO) in a 100 mm culture dish for 24 hours. Subsequently, the medium was replaced with medium supplemented with 10% Exo-depleted FBS. Exo-depleted FBS was obtained by ultracentrifugation at 100,000 x g for 18 hours. After 72 hours of culture, the culture medium was collected and centrifuged at 300 x g for 10 minutes, 2,500 x g for 25 minutes, and 10,000 x g for 1 hour at 4°C. The supernatant was filtered using a 0.22 μm filter and ultracentrifuged at 100,000 x g for 2 hours. Subsequently, the Exo pellet was washed by centrifugation at 100,000 x g for 2 hours with sterile PBS and then 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. Subsequently, the medium was replaced with medium supplemented with 10% Exo-depleted FBS. After 72 hours, the culture supernatant was ultracentrifuged to isolate Exo and stored at -80°C until use (concentrated-CM; concentrated CM). Concentrated CM derived from GW4869-treated MSCs (Exo secretion suppression) was used as a vehicle control group for all mouse experiments. The amount of Exo was determined using a BCA assay kit (Thermo Fisher Scientific, Waltham, MA) for the measurement of total protein. The Exo size and number were determined using qNano (Izon Science, Christchurch, New Zealand) and transmission electron microscopy (TEM; Jeol, Tokyo, Japan) based on the manufacturer's instructions.

[0062] For Western blot analysis, WJ-MSCs lysates and Exos were lysed in RIPA buffer (Thermo Fisher) with protease inhibitors. Equal amounts of protein were applied to SDS-PAGE and analyzed with primary antibodies against 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).

[0063] As a result, transmission electron microscopy analysis showed that the 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, while the cytoplasmic marker β-actin was not expressed in any of the exosomes (Figure 2B). The results of qNano analysis showed that the average particle size of the exosomes was 110 - 120 nm for both CTL-Exo and TSG-Exo (Figure 2C). The production of TSG-primed exosomes derived from WJ-MSCs was 50% higher than that of naive WJ-MSC-derived exosomes (Figures 2C, D). TSG-Exo had significantly higher expression levels of IL-10, TGFβ, COX2, and IDO compared to CTL-Exo, but had lower expression of pro-inflammatory cytokines including IFNγ, TNFα, and IL-1β (Figure 3E). Consistent with the mRNA expression results, the protein expression levels of TGFβ, COX2, and IDO were significantly increased in TSG-Exo (Figure 3F).

[0064] [Example 2] Confirmation of the immunomodulatory effect of TSG-primed exosomes - Suppression of T cell proliferation To evaluate the immunomodulatory ability of TSG-primed exosomes, the expression levels of anti-inflammatory cytokines and pro-inflammatory cytokines were measured in activated mononuclear cells (MNCs).

[0065] Total RNA was extracted from cells, Exos, or tissue samples using TRIzol reagent (Invitrogen, Waltham, MA) for quantitative RT-PCR, and cDNA was synthesized from total RNA using Superscript TM reverse transcriptase (Invitrogen). Quantitative PCR was measured using SYBR Green Master Mix (Applied Biosystems, Foster City, CA) with an MX300P thermal cycler (Stratagene, San Diego, CA). The expression levels of mRNA were normalized to the levels of GAPDH. The primer sequences for qRT-PCR were as described in Table 1 below.

[0066]

Table 1

[0067] Mononuclear cells (MNCs) for apoptosis analysis were separated from hUCB using Ficoll-Hypaque (GE Healthcare, Chicago, IL) gradient centrifugation based on Non-Patent Document 2 and resuspended in RPMI 1640 medium supplemented with 10% Exo-depleted FBS. Umbilical cord blood (UCB) units were obtained from the Korean Catholic Hematopoietic Stem Cell Bank (CHSCB) from April 2019 to June 2020 with the approval of the Institutional Review Board (IRB No. 2019-0467-0003). MNCs were stimulated with 5 μg / ml Con A (Concanavalin A) and seeded in 96-well plates (1×10 5 / well) in growth medium with or without naive or TSG-primed WJ-MSC-derived Exos (4 μg) for 72 hours. Annexin V / 7AAD (BD Biosciences, Franklin Lakes, NJ) staining was performed, and apoptosis of the cells was determined based on the manufacturer's instructions and analyzed by flow cytometry.

[0068] 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 provided with the approval of the Institutional Review Board of the Catholic University (IRB No. 2019-2891-0003) from the Korean Red Cross (Seoul, Korea). PBMCs were separated by centrifugation through a Ficoll-Hypaque density gradient and resuspended in RPMI 1640 medium supplemented with 10% Exo-depleted FBS.

[0069] T cell proliferation was quantified by CFSE dilution. The isolated PBMCs were labeled with 2 μM CFSE (CellTrace CFSE Cell Proliferation Kit, Thermo Fisher Scientific). CFSE-labeled PBMCs (1×10 5 / well) were co-cultured in 96-well plates in the presence or absence of naive or TSG-primed WJ-MSC-derived Exos (4 μg) in the presence of anti-CD3 / CD28 microbeads (Gibco) and recombinant human IL-2 (30 U / ml, Peprotech). After 6 days of culture, the cells were stained with human monoclonal antibodies fluorescently labeled with CD45, CD3, CD4, or CD8 (BD Biosciences) and measured by flow cytometry. All cells were gated on 7AAD-negative cells.

[0070] As a result, when MNCs stimulated with Con A (concanavalin A) were treated with exosomes, the expression levels of COX2, NOS2, IDO, IL-10, and TGFβ increased, while the expression levels of TNFα and IL-1β decreased. This was more prominent in activated MNCs treated with TSG-Exo than in those treated with CTL-Exo (Figure 3G). TSG-Exo slightly decreased the 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 more potently suppressed the proliferation of total T, CD4+T, and CD8+T cells than CTL-Exo (Figures 4A, B). The proportion of cells in the initial division cycle 1 increased significantly in PBMCs treated with TSG-Exo, and the proportions of cells in division cycles 4 and 5 decreased significantly (Figure 4C). Such results suggest that TSG-primed exosomes increase the production of anti-inflammatory cytokines and have a significant inhibitory effect on T cell proliferation.

[0071] [Example 3] Confirmation of the immunomodulatory effect of TSG-primed exosomes - improvement of regulatory T cell and M2 macrophage polarization <3-1>Confirmation of T helper cell (Th) differentiation of TSG-primed exosomes It has been reported that MSC exosomes can mediate immunomodulatory effects through the differentiation of regulatory T cells (Tregs). Therefore, the present inventors examined the differentiation of T helper cells (Th) to confirm the effect of TSG-Exo on specific subsets of T cells.

[0072] Th cells were induced by stimulating family-specific cytokines on CD4+ cells purified from human PBMCs in the presence of exosomes. Specifically, the separation of CD4+ T cells from PBMC lymphocytes was performed using 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 specific cytokines were added for the differentiation of specific Th cells: IFNγ (ml) and IL-12 (25 ng / ml) for Th1; IL-6 (50 g / ml) and TGFβ (g / ml) for Th17; TGFβ (g / ml) and retinoic acid (10 nM) for Treg. Cytokine-treated CD4+ T cells (5×10 5 / well) were added with naive or TSG-primed WJ-MSC-derived Exos in 24-well plates and cultured for 5 days. For intracellular staining, cells were stimulated with a cell stimulation cocktail (Invitrogen) for 5 hours before staining. After staining the cells with anti-CD3, anti-CD4, and anti-CD25 antibodies, which are cell surface markers, the cells were fixed / permeabilized and stained with fluorescently labeled anti-IFNγ, anti-IL-17A, and anti-Foxp3 antibodies (BD Biosciences). The stained cells were analyzed by flow cytometry.

[0073] As a result, co-culture with TSG-Exo significantly decreased the induction of IFNγ+CD4+ Th1 and IL-17A+CD4+ Th17 cells compared to CTL-Exo and increased Foxp3+CD25+CD4+ Treg cells (Figures 5D and 6). M1 macrophages showed a round morphology, while M2 macrophages showed a spindle-shaped morphology (Figure 7).

[0074] <3-2>Confirmation of enhanced M2 macrophage polarization of TSG-primed exosomes It has been reported that MSC exosomes can mediate immunomodulatory effects through the induction of M2 macrophage polarization. Therefore, the present inventors examined whether TSG-Exo can regulate the phenotype of macrophages in order to confirm the effect of TSG-Exo on specific subsets of T cells.

[0075] GM-CSF (in the case of M1 type) or M-CSF (in the case of M2 type)-stimulated primary human macrophages were cultured with exosomes in the presence of M1-type cytokines or M2-type cytokines. Specifically, CD14+ monocytes were isolated from hUCB-derived MNCs using CD14-binding microbeads (Miltenyi Biotec). To generate macrophages, monocytes (5×10 5 / well) were cultured with GM-CSF (50 ng / ml; Peprotech) or M-CSF (100 ng / ml) in RPMI 1640 medium (Gibco) containing 10% FBS for 6 days. For M1 polarization in 24-well plates, GM-CSF-derived macrophages were stimulated with IFNγ ng / ml) + LPS (1 μg / ml) together with naive or TSG-primed WJ-MSC-derived Exo for 48 hours. For M2 polarization, M-CSF-derived macrophages were stimulated with IL-4 (20 ng / ml) and IL-13 (20 ng / ml) together with naive or TSG-primed WJ-MSC-derived Exo for 48 hours. After co-culture, the cells were stained with human monoclonal antibodies fluorescently labeled with CD14, CD80, CD86, CD206, and CD163 (BD Biosciences) and analyzed by flow cytometry.

[0076] As a result, the M1-type cell surface markers CD80+ and CD86+ were significantly decreased in M1-type macrophages cultured with TSG-Exo, whereas they were cultured with CTL-Exo. However, the expression of the 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 (Figure 5E and Figure 8). Such results indicate that TSG-Ex It is suggested that o more effectively induces the M2 macrophage phenotype than naive exosomes. Therefore, TSG treatment of MSCs can generate exosomes with improved immunomodulatory properties.

[0077] [Example 4] Confirmation of the colitis remission effect induced by DSS of TSG-primed exosomes The inventors confirmed the potential protective effect of TSG-Exo in a colitis mouse model induced by 3% DSS (Figure 9A).

[0078] Specifically, for the production of a colitis mouse model induced by DSS, 3% (w / v) DSS (Dextran sulfate) was added to the drinking water of 7-week-old C57BL / 6 mice for 7 days. It was induced by administering sodium; MP Biomedicals, Santa Ana, CA). The mice were divided into four groups of 10 each according to the group: (1) control group (negative control group), (2) DSS administered with concentrated CM (solvent control group, 200 μl) extracted from GW4869-treated MSCs (positive control group), (3) DSS administered with Exos derived from naive MSCs, (4) DSS administered with Exos derived from TSG-primed MSCs. Groups (3) and (4) were intraperitoneally injected with 200 μg of Exos diluted in 200 μl of PBS into the mice on days 1, 3, and 5. The mice were euthanized on day 10. The severity of colitis was evaluated daily using a disease activity index (DAI) including weight loss (0 - 4), stool consistency (0 - 4), stool blood (0 - 4), coat roughness (0 - 4), rectal prolapse (0 - 3), hunched posture (0 - 3), bedding soiling (0 - 2), not inquisitive / alert (0 - 2). All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Catholic University (IACUC no. 2019 - 0301 - 03). As a solvent control group, the culture supernatant of MSCs treated with GW4869 (10 μM), an inhibitor of nSMase2 (neutral sphingomyelinase supernatant 2) that regulates exosome secretion (GW - CM), was used.

[0079] The length of the colon of the sacrificed mice on day 10 was measured. The colon tissues were fixed in 4% formaldehyde (Wako, Osaka, Japan), embedded in paraffin, and then sectioned into 5 - μm slices. For histological analysis, the colon sections were stained with hematoxylin & eosin (H&E). The histopathological score was determined in a blinded manner based on the degree of inflammatory cell infiltration (0 - 4) and the degree of tissue damage (0 - 4).

[0080] The infiltration of neutrophils into the colon tissue was measured using the Myeloperoxidase (MPO) Colorimetric Activity Assay Kit (Sigma). The colon was homogenized in MPO assay buffer, and the supernatant was collected by centrifugation at 13,000 x g for 10 minutes. The supernatant was mixed with MPO assay buffer and MPO substrate, cultured at 25 °C for 120 minutes, and then the TNB (tetramethylbenzidine) probe was added. Absorbance was measured at 412 nm using a spectrophotometer (Biotek, Winooski, VT).

[0081] As a result, GW4869 successfully inhibited exosome release in MSCs. Colitis mice treated with GW-CM showed a significantly increased disease activity index (DAI) such as persistent weight loss, stool hardness, bloody diarrhea, and general activity. However, exosome treatment improved weight loss and DAI, especially in the TSG-Exo treatment group (Figures 9B, C). Furthermore, the shortening of the colon length due to DSS administration was significantly improved in TSG-Exo-treated mice compared to CTL-Exo-treated mice (Figures 10D, E). Histological examination showed that TSG-Exo-treated mice maintained colon tissue integrity; there was significant structural disruption, loss of crypt cells, and reduced infiltration of inflammatory cells; and they had a lower histological score compared to CTL-Exo-treated mice (Figures 10F, G). Furthermore, the MPO (myeloperoxidase) activity indicating neutrophil infiltration in mice sacrificed on day 10 was significantly decreased in TSG-Exo-treated mice compared to CTL-Exo-treated mice (Figure 10H). Such results indicate that TSG-Exo has improved protective activity against intestinal inflammation in DSS-induced colitis compared to naive exosomes.

[0082] [Example 5] Confirmation of the effect of TSG-primed exosomes on improving the polarization of regulatory T cells and M2 macrophages in the inflamed colon Immune cell responses play an important role in the etiology of inflammatory bowel disease (IBD). In particular, the balance between Tregs and other T cells in the intestinal microenvironment plays an important role in the remission of colitis, and macrophages mediate the inflammatory response through M1 / M2 polarization in the colon and play an important role in the pathogenesis of IBD. Therefore, we attempted to confirm the immunomodulatory effect of TSG-primed exosomes on the polarization of regulatory T cells and M2 macrophages in the inflamed colon. Next, the inventors examined the effect of TSG-primed exosomes on the immune cell profile of DSS-induced colitis mice.

[0083] Specifically, 3%DSS-colitis mice (n = 2-4 mice per group) were sacrificed on day 10, and after obtaining colon tissues, the levels of Th1, Th2, Th17, and Treg cells in the colon tissues were evaluated. Lamina propria cells were isolated from the intestine. The colon tissues were cut into small pieces (3 mm × 3 mm) and cultured with HBSS buffer containing 2 mM EDTA (Sigma) in a shaking incubator at 220 rpm for 30 minutes. After removing the epithelium at 37°C, the tissues were digested in RPMI containing 5% FBS, 1 mg / ml of Collagenase D (Sigma), and 0.1 mg / ml of DNase I (Sigma) in a shaking incubator at 220 rpm at 37°C for 60 minutes. The digested samples were filtered through a 40-μm cell strainer and then centrifuged at 300 xg for 5 minutes. The isolated cells were stained with fluorescently labeled antibodies against CD11b, F4 / 80, CD206, and arginase 1 (BD Biosciences) and analyzed by flow cytometry.

[0084] As a result, the expression levels of Th1 (T-bet), Th2 (GATA3) and Th17 (RORγ family transcription factors) were increased in colon tissues of GW-CM-treated mice, but decreased in exosome-treated mice. Notably, GATA3 expression was further significantly decreased in TSG-Exo-treated mice compared to CTL-Exo-treated mice. Foxp3 and Treg transcription factors were significantly increased in TSG-Exo-treated mice compared to CTL-Exo-treated mice (Figure 11A).

[0085] In addition, the 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. The M2-type marker expression of Arg1 and CD206 was increased in exosome treatment, and this effect was significantly greater in the TSG-Exo-treated group (Figure 11B).

[0086] The percentage of F4 / 80+CD11b+ cells (macrophages) in the lamina propria of the colon was significantly increased in DSS-induced colitis mice compared to negative control mice (Figure 12C, D). Exosome treatment did not affect the percentage of F4 / 80+CD11b+ cells. Although TSG-primed exosome-treated mice had no effect on mucosal inflammation (Fig. 12C, D), the expression levels of CD206 and arginase 1 in colonic macrophages were significantly elevated 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.

[0087] [Example 6] ER stress-induced WJ-MSC or exosome analysis <6-1> Identification of ER stress-induced WJ-MSC characteristics To investigate the effect of endoplasmic reticulum stress (ER-stress) on the immunomodulatory properties of human MSCs, the ER-stress inducers thapsigargin (TSG, Th, apsigargin), tunicamycin (Tu), brefeldin A (BFA), dithiothreitol (DTT, dithiothreitol), or MG132 (MG) were each treated in the same manner as in Example <1-2>, and the expression of ER stress markers and inflammatory genes was confirmed in WJ-MSCs induced with ER stress.

[0088] As a result, as shown in Figure 13, the ER-stress markers GRP94 and CHOP were significantly increased in human MSCs after treatment with ER-stress inducers. In addition, MSCs treated with ER-stress inducers had significantly higher expression levels of anti-inflammatory cytokines COX2, IDO, and IL-10 than untreated MSCs, but it was confirmed that the expression of pro-inflammatory cytokines including IL-1β, IFNγ, and TNFα was even lower.

[0089] <6-2>Confirmation of the immunomodulatory effect of ER-stress-induced WJ-MSCs - Inhibition of T cell proliferation To evaluate the immunomodulatory properties of MSCs treated with an endoplasmic reticulum stress (ER-stress) inducer on T cell proliferation, PBMCs and MSCs were co-cultured in the same manner as in Example <1-2>.

[0090] As a result, as shown in Figure 14, it was confirmed that MSCs treated with ER-stress inducers (TSG, Tu, BFA, DTT, MG) had an even greater inhibitory effect on the proliferation of total T, CD4+, and CD8+ T cells compared to untreated MSCs.

[0091] These results indicate that MSCs subjected to ER stress by ER-stress inducers exhibit a function that is effective in increasing the production of anti-inflammatory cytokines and strengthening the inhibition of T cell proliferation.

[0092] <6-3>Confirmation of the Immunomodulatory Effect of Exosomes Secreted from ER Stress-Induced WJ-MSCs - Inhibition of T Cell Proliferation To confirm the immunosuppressive effect of exosomes secreted from MSCs treated with ER stress inducers (TSG, Tu, BFA, DTT, MG), PBMCs and exosomes were co-cultured in the same manner as in [Example 2].

[0093] As a result, as shown in Figure 15, it was confirmed that exosomes treated with TSG, BFA, and Tu more potently inhibited the proliferation of total T, CD4+ T, and CD8+ T cells compared to the control group (cont-exo).

[0094] These results indicate that ER-stress inducers can generate exosomes with improved immunomodulatory properties in MSCs.

[0095] [Statistical Analysis] Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test with GraphPad Prism v8.0.1 (GraphPad Software, San Diego, CA). All data were presented as mean ± S.D. A P-value < 0.05 was considered statistically significant. [Industrial Applicability]

[0096] Exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer (TSG-Exo) according to the present invention increased the levels of anti-inflammatory cytokines, regulatory T cells, and M2 macrophages, and decreased the levels of pro-inflammatory cytokines, helper T cells, and M1 macrophages, etc., increasing the immunomodulatory ability and effectively alleviating inflammation in a colitis mouse model. Therefore, the present invention can be effectively used as a composition for preventing or treating inflammatory diseases and a composition for immunomodulation, and thus has industrial applicability. [Sequence Listing Free-Text]

[0097] Array Catalog Free Text Array number 1 corresponds to the forward primer sequence of hCOX-2. Array number 2 corresponds to the reverse primer sequence of hCOX-2. Array number 3 corresponds to the forward primer sequence of hIDO. Array number 4 corresponds to the reverse primer sequence of hIDO. Array number 5 corresponds to the forward primer sequence of hIFNγ. Array number 6 corresponds to the reverse primer sequence of hIFNγ. Array number 7 corresponds to the forward primer sequence of hIL-10. Array number 8 corresponds to the reverse primer sequence of hIL-10. Array number 9 corresponds to the forward primer sequence of hIL-1β. Array number 10 corresponds to the reverse primer sequence of hIL-1β. Array number 11 corresponds to the forward primer sequence of hNOS2. Array number 12 corresponds to the reverse primer sequence of hNOS2. Array number 13 corresponds to the forward primer sequence of hTGFβ. Array number 14 corresponds to the reverse primer sequence of hTGFβ. Array number 15 corresponds to the forward primer sequence of hTNFα. Array number 16 corresponds to the reverse primer sequence of hTNFα. Array number 17 corresponds to the forward primer sequence of mArg1. Array number 18 corresponds to the reverse primer sequence of mArg1. Array number 19 corresponds to the forward primer sequence of mCD206. Array number 20 corresponds to the reverse primer sequence of mCD206. Array number 21 corresponds to the forward primer sequence of mCXCL9. Array number 22 corresponds to the reverse primer sequence of mCXCL9. Array number 23 corresponds to the forward primer sequence of mFoxp3. Array number 24 corresponds to the reverse primer sequence of mFoxp3. Array number 25 corresponds to the forward primer sequence of mGATA3. Array number 26 corresponds to the reverse primer sequence of mGATA3. Array number 27 corresponds to the forward primer sequence of miNOS. Array number 28 corresponds to the reverse primer sequence of miNOS. Array number 29 corresponds to the forward primer sequence of mMCP1. Array number 30 corresponds to the reverse primer sequence of mMCP1. Array number 31 corresponds to the forward primer sequence of mRORγ. Array number 32 corresponds to the reverse primer sequence of mRORγ. Array number 33 corresponds to the forward primer sequence of mT-bet. Array number 34 corresponds to the reverse primer sequence of mT-bet.

Claims

1. A method for promoting the production of exosomes derived from mesenchymal stem cells, comprising the step of treating mesenchymal stem cells collected from humans with an endoplasmic reticulum stress inducer, wherein the endoplasmic reticulum stress inducer is thapsigargin (TSG).

2. The method according to claim 1, wherein the mesenchymal stem cells are mesenchymal stem cells obtained from any one or more tissues selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, and fetal liver.

3. The method according to claim 1, wherein the size of the exosomes is 10 to 200 nm.

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