WO2015172659A1

WO2015172659A1 - Uses of il-17 in enhancing immune-suppression function of mesenchymal stem cells

Info

Publication number: WO2015172659A1
Application number: PCT/CN2015/077862
Authority: WO
Inventors: 时玉舫; 韩晓燕; 王莹
Original assignee: 中国科学院上海生命科学研究院
Priority date: 2014-05-14
Filing date: 2015-04-29
Publication date: 2015-11-19
Also published as: CN105079792A

Abstract

Provided are uses of IL-17 in enhancing an immune-suppression function of mesenchymal stem cells. In particular, the present invention provides uses of an interleukin-17, a derivative of interleukin-17 or an agonist thereof for preparing a preparation or kit for enhancing an immune-suppression function of the mesenchymal stem cells; up-regulating the expression of imunosuppressive factors in the mesenchymal stem cells; enhancing the stability of mRNAs of the immunosuppressive factors; reducing the expression level of RNA-binding protein AUF1; inhibiting the proliferation of T cells; and treating hepatitis or liver damage.

Description

Application of IL-17 in improving the immunosuppressive function of mesenchymal stem cells

Technical field

The present invention belongs to the field of biomedicine, and in particular, the present invention relates to the use of IL-17 for enhancing the immunosuppressive function of mesenchymal stem cells.

Background technique

Mesenchymal stem cells (MSC) are important members of the stem cell family. They are derived from the mesoderm and ectoderm in early development and belong to pluripotent stem cells. MSCs are originally found in the bone marrow because of their multi-directional differentiation potential and hematopoiesis. Supporting and promoting stem cell implantation, immune regulation and self-replication are increasingly attracting attention. For example, mesenchymal stem cells can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, heart muscle, endothelium, etc. after continuous induction and cryopreservation. It still has multi-directional differentiation potential and can be used as an ideal seed cell for the repair of tissue and organ damage caused by aging and lesions.

The immunosuppressive mechanism of MSCs for T cells has been extensively studied both in vivo and in vitro. It is generally believed that MSCs inhibit T cells by not inducing T cell apoptosis, but in the cell cycle of T cells. Stay in G0/G1 period. Studies have also shown that MSC can promote the apoptosis of activated T cells, but can promote the survival of T cells in resting state, which also indicates that MSC exerts immunosuppression depending on the stimulation of inflammatory cytokines released by T cell activation.

Interleukin-17A (interleukin 17A, IL-17A, interleukin-17, abbreviated as IL-17) is one of more than 30 kinds of interleukins that have been discovered so far, ranking 17th according to the serial number. IL-17 is secreted by CD4+ T cells and can induce the synthesis and secretion of IL-6, IL-8, G-CSF and PGE2 by epithelial cells, endothelial cells and fibroblasts, and promote the expression of ICAM-1. In recent years, IL-17 has been found to be a proinflammatory cytokine mainly produced by activated T cells, which can promote the activation of T cells and stimulate the production of various cytokines such as IL-6 and IL in epithelial cells, endothelial cells and fibroblasts. -8, granulocyte-macrophage stimulating factor (GM-CSF) and chemical stimulating hormone 1 and cellular adhesion molecule 1 (CAM-1), resulting in inflammation. IL-17 is an early promoter of T cell-induced inflammatory responses that amplify the inflammatory response by promoting the release of pro-inflammatory cytokines. After binding to the receptor, IL-17 exerts its biological effects through the MAP kinase pathway and the nuclear factor kB (NF-kB) pathway. Th17 cells secrete IL-17A, IL-17F, IL-6, and tumor necrosis factora (TNF-α). These cytokines can collectively mobilize, recruit, and activate neutrophils. IL-17 produced by Th17 cells can effectively mediate the excitatory process of neutrophil mobilization, thereby effectively mediating the inflammatory response of tissues.

As a pro-inflammatory cytokine, IL-17 can act on a variety of cell types, thereby promoting the expression of other cytokines, chemokines and metalloproteinases, including TNFα, IL-1β, IL-6, GM-CSF, G-CSF, CXCL1, MMP-3, and the like. At the same time, IL-17 can also promote the expression of multiple target genes in combination with other cytokines such as TNFα. In vivo experimental studies have revealed the important role of IL-17 family cytokines in the body's antimicrobial infection. Although IL-17 protects against bacterial and fungal infections in the body, over-activation of the IL-17 signaling pathway leads to autoimmune diseases. In human autoimmune diseases, IL-17 is significantly elevated, and many studies have shown that IL-17 is involved in the pathogenesis of many autoimmune diseases, including MS (multiple sclerosis), RA (rheumatoid arthritis), and IBD ( Inflammatory bowel disease).

Summary of the invention

It is an object of the present invention to provide use of interleukin-17, a derivative of interleukin-17 or an agonist thereof.

Another object of the invention is to provide the use of an interleukin-17 antagonist.

Another object of the present invention is to provide an IL-17-treated mesenchymal stem which has improved immunosuppressive ability. cell.

In a first aspect of the invention, there is provided a use of an interleukin-17, a derivative of interleukin-17 or an agonist thereof for the preparation of a formulation or kit for:

(1) improving the immunosuppressive function of mesenchymal stem cells; and/or

(2) up-regulating the expression of immunosuppressive factors in MSC; and/or

(3) enhancing the stability of mRNA of immunosuppressive factors in MSC; and/or

(4) reducing the expression level of the RNA binding protein AUF1; and/or

(5) inhibiting the proliferation of T cells; and/or

(6) Treatment of hepatitis or liver damage.

In another preferred embodiment, the interleukin-17 is a mammalian interleukin-17.

In another preferred embodiment, the interleukin-17 is human interleukin-17.

In another preferred embodiment, the amino acid sequence of interleukin-17 is as shown in SEQ ID NO.: 1.

In another preferred embodiment, the interleukin-17 derivative comprises a modified interleukin-17 molecule, a protein molecule having an amino acid sequence homologous to native interleukin-17 and having natural interleukin-17 activity, and dimerization of interleukin-17. A body or multimer, a fusion protein containing an interleukin-17 amino acid sequence.

In another preferred embodiment, the modified interleukin-17 molecule is PEGylated interleukin-17.

In another preferred embodiment, the "protein molecule having an amino acid sequence homologous to natural interleukin-17 and having natural interleukin-17 activity" means that the amino acid sequence thereof has ≥85% as compared with SEQ ID NO.: Derived, preferably > 90% homology, more preferably > 95% homology, optimally > 98% homology; and a protein molecule having native interleukin-17 activity.

In another preferred embodiment, the "immunosuppressive function of mesenchymal stem cells" refers to an immunosuppressive function of mesenchymal stem cells to T cells.

In another preferred embodiment, the formulation or kit further comprises IFNy and/or TNF[alpha].

In another preferred embodiment, the agonist means a substance capable of increasing the activity and/or content of interleukin-17 or a derivative thereof in vivo or in vitro. The substance may be a synthetic or natural compound, protein, nucleotide or the like.

In another preferred embodiment, the MSC cells are bone marrow derived MSC cells, umbilical cord derived MSC cells, adipose derived MSC cells, placental derived MSC cells, and/or pulp derived MSC cells.

In another preferred embodiment, the hepatitis comprises autoimmune hepatitis, viral hepatitis; the liver damage includes alcoholic liver damage, drug-induced liver damage, liver fibrosis, cirrhosis.

In another preferred embodiment, the immunosuppressive factors include iNOS, indole-2,3-dioxygenase, PGE2, TSG6, IL-6, HO-1, IL-10, PD-L1, Galetin, and / or B7-H4.

In a second aspect of the invention, there is provided a use of an interleukin-17 antagonist for the preparation of a formulation or kit for:

(1) reducing the immunosuppressive function of mesenchymal stem cells; and/or

(2) down-regulating the expression of immunosuppressive factors in MSC; and/or

(3) reducing the stability of mRNA of immunosuppressive factors in MSC; and/or

(4) enhancing the expression level of the RNA binding protein AUF1; and/or

(5) Promote the proliferation of T cells.

In another preferred embodiment, the antagonist of interleukin-17 refers to a substance capable of reducing the activity of interleukin-17 in vivo or in vitro.

In another preferred embodiment, the antagonist of interleukin-17 may be a small miRNA, an anti-interleukin-17 antibody or an iNOS inhibitor, an Act1 protein inhibitor, an AUF1 protein agonist, an interleukin-17 receptor inhibitor, NFκB. Inhibitor, TRAF6 inhibitor.

In another preferred embodiment, the formulation comprises a pharmaceutical composition, a nutraceutical composition, a food composition, or an experimental agent.

In a third aspect of the invention, there is provided a composition comprising interleukin-17 or a derivative thereof, IFNy and TNFa.

In another preferred embodiment, the molar ratio of the interleukin-17 or its derivative, IFNγ and TNFα is from 1-10:10:10.

In another preferred embodiment, the composition comprises a solid formulation, a liquid formulation, preferably in the form of a dry powder or solution.

In another preferred embodiment, the composition is a pharmaceutical composition.

In a fourth aspect of the invention, there is provided an isolated population of MSC cells consisting of or consisting essentially of MSC cells, and said MSC cells having enhanced ability to inhibit T cell proliferation,

And, the MSC cells are selected from the group consisting of:

(1) an in vitro pretreated MSC cell population, wherein the pretreatment refers to simultaneous, sequential or sequential treatment with (i) interleukin-17 or a derivative thereof, (ii) IFNγ and (iii) TNFα;

(2) a cell population in which Act1 protein is overexpressed and/or AUF1 protein is deleted or reduced in activity;

(3) an in vitro pretreated MSC cell population, wherein the pretreatment refers to treatment with IFNγ and TNFα;

(4) A combination of group (1), group (2), and group (3).

In another preferred embodiment, in the pretreatment, the conditions are as follows: 0.5-1000 ng/ml (preferably 1-200 ng/ml, more preferably 1-20 ng/ml, optimally 1-10 ng/ml) IFNγ, 0.5-1000 ng/ml (preferably 1-200 ng/ml, more preferably 1-20 ng/ml, optimally 1-10 ng/ml) of TNFα, and 0.2-1000 ng/ml (preferably 0.5-) 200 ng/ml, more preferably 1-20 ng/ml, optimally 1-10 ng/ml) interleukin-17.

In another preferred embodiment, 1-2 ng/ml IFNγ, 1-2 ng/ml TNFα, and 0.5-2 ng/ml interleukin-17 are used in the pretreatment; or 10 ng/ml IFNγ, 10 ng/ml TNFα, and 10 ng are used. /ml IL-17.

In another preferred embodiment, the enhanced ability to inhibit T cell proliferation refers to I1/I0 ≥ 1.5 (preferably ≥ 2, more preferably ≥ 4), wherein I1 is the proliferation of T cells by the MSC cells. Percent inhibition rate; while I0 is the percent inhibition of T cell proliferation by wild-type MSC cells of the same species in the control group.

In another preferred embodiment, said "consisting essentially of" means that the MSC cells comprise at least 90%, preferably at least 95%, more preferably at least 99% of the total number of cells in said population of cells.

In another preferred embodiment, the MSC cell population has the characteristic that, after administration of the MSC cell population to an animal, a change in the animal from the group of the following occurs:

(a) a decrease in the level of serum ALT;

(b) a decrease in mononuclear cells in the liver;

(c) a decrease in the number of CD4 ⁺ T cells; and/or

(d) Decreased CD8 ⁺ T cell count.

In another preferred example,

(a) serum ALT levels are reduced by 50% to 80%;

(b) Mononuclear cells in the liver are reduced by 50% to 80%;

(c) a decrease in the number of CD4 ⁺ T cells by 50% to 80%; and/or

(d) The number of CD8 ⁺ T cells decreased by 50% to 75%.

In another preferred embodiment, the preprocessing process includes the steps of:

After trypsinizing the MSC cells, the cells are cultured until the cells are attached, and then the cytokines at the above concentrations are added, and after further culture for 6-24 hours, the cell population is further digested and collected.

In another preferred embodiment, the MSC cells are bone marrow-derived MSC cells, umbilical cord-derived MSC cells, adipose-derived MSC cells, placental-derived MSC cells, and/or pulp-derived MSC cells.

In a fifth aspect of the invention, there is provided a genetically engineered cell strain which is genetically engineered to result in overexpression of an endogenous Actl protein and/or a decrease in AUF1 protein or activity.

In another preferred embodiment, the cell strain is a mammalian MSC cell line.

In another preferred embodiment, the cell strain is for enhancing the immunosuppressive function of mesenchymal stem cells.

In a sixth aspect of the invention, an isolated protein complex is provided, the protein complex being a protein complex bound by an Act1 protein and an AUF1 protein.

In another preferred embodiment, the protein complex has a molecular weight of from 80 KD to 130 KD.

A seventh aspect of the invention provides the use of the protein complex of the sixth aspect of the invention for screening a drug or a compound which promotes or inhibits the formation of the Act1 protein and the AUF1 protein Complex.

In another preferred embodiment, the medicament is for:

(1) improving the immunosuppressive function of mesenchymal stem cells; and/or

(2) up-regulating the expression of immunosuppressive factors in MSC; and/or

(3) enhancing the stability of mRNA of immunosuppressive factors in MSC; and/or

(4) reducing the expression level of the RNA binding protein AUF1; and/or

(5) inhibiting the proliferation of T cells; and/or

(6) Treatment of hepatitis or liver damage.

In another preferred embodiment, when the protein complex is used to screen for a drug, the application comprises the steps of:

(a) co-cultivating the target substance with MSC cells;

(b) detecting the content of the protein complex in the cultured cells. When the protein complex content in the cell is higher than the blank control, the substance is a positive candidate substance.

In another preferred embodiment, the screening further comprises a positive control group, preferably IL17 is added to the positive control group. (Experiments have shown that IL17 enhances the ability of two proteins to form a complex.)

In an eighth aspect of the invention, a kit is provided, the kit comprising the following components:

(a) interleukin-17 or a derivative thereof;

(b) IFNγ; and

(c) TNFα;

And instructions for use,

Wherein the components (a), (b) and (c) are respectively located in one or more different containers or in the same container.

In another preferred embodiment, the description describes: the kit is for

(1) improving the immunosuppressive function of mesenchymal stem cells; and/or

(2) up-regulating the expression of immunosuppressive factors in MSC; and/or

(3) enhancing the stability of mRNA of immunosuppressive factors in MSC; and/or

(4) reducing the expression level of the RNA binding protein AUF1; and/or

(5) inhibiting the proliferation of T cells.

According to a ninth aspect of the invention, there is provided a kit comprising the pharmaceutical composition according to claim 3 and instructions, wherein the pharmaceutical composition is described for:

(1) improving the immunosuppressive function of mesenchymal stem cells; and/or

(2) up-regulating the expression of immunosuppressive factors in MSC; and/or

(3) enhancing the stability of mRNA of an immunosuppressive factor; and/or

(4) reducing the expression level of the RNA binding protein AUF1; and/or

(5) inhibiting the proliferation of T cells; and/or

(6) Treatment of hepatitis or liver damage.

In a tenth aspect of the invention, a method of treating hepatitis or liver damage is provided, the method comprising the steps of:

A therapeutically effective amount of MSC cells is administered to the subject in need thereof.

In another preferred embodiment, the liver damage is cirrhosis. In another preferred embodiment, the subject is a mammal (e.g., a human).

In another preferred embodiment, the application is an intravenous infusion.

In another preferred embodiment, the MSC cell is the MSC cell population of the fourth aspect of the invention.

In another preferred embodiment, the MSC cell is a pre-treated MSC cell population, wherein the pretreatment refers to (i) interleukin-17 or a derivative thereof, (ii) IFNγ, and (iii) TNFα simultaneously. Process in sequence or sequentially.

In another preferred embodiment, the MSC cells are in vitro pretreated MSC cell populations, wherein the pretreatment refers to simultaneous, sequential or sequential treatment with IFNγ and TNFα.

In another preferred embodiment, the density of the MSC cell population during the pretreatment is 1×10 ⁴ cells/ml-5×10 ⁶ cells/ml, preferably 5×10 ⁴ cells/ml-5×10 ⁵ cells. /ml.

In another preferred embodiment, the concentration of interleukin-17 during the pretreatment is from 0.5 to 1000 ng/ml, preferably from 1 to 100 ng/ml.

In another preferred embodiment, the concentration of IFNγ during the pretreatment is from 0.5 to 1000 ng/ml, preferably from 1 to 100 ng/ml.

In another preferred embodiment, the concentration of TNFα in the pretreatment is from 0.5 to 1000 ng/ml, preferably from 1 to 100 ng/ml.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

1 includes FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H showing that IL-17 enhances the immunosuppressive function of MSCs.

Figure 2, comprising Figures 2A and 2B, shows that IL-17 enhances the immunosuppressive properties of MSCs depending on the activity of iNOS.

Figure 3 shows that IL-17 enhances the immunosuppressive function of MSCs independent of the effects on T cell apoptosis.

Figure 4, comprising Figures 4A, 4B, 4C, 4D, 4E, 4F, and 4G, shows that IL-17 synergistic inflammatory cytokines up-regulate the expression of iNOS in MSCs.

Figure 5, comprising Figures 5A, 5B, 5C, and 5D, shows that IL-17 plays an important role in enhancing immunosuppression and gene expression in MSCs requiring Act1 involvement.

Figure 6 includes Figures 6A, 6B, and 6C showing that IL-17, IFNy, and TNF[alpha] pretreated MSCs are effective in treating ConA-induced liver damage.

Figure 7 includes Figures 7A, 7B, 7C, and 7D showing that the therapeutic effects of IL-17, IFNγ, and TNFα pretreated MSCs on CIH depend on their immunosuppressive function on T cells without affecting T cell subsets. proportion.

Panels A-E in Figure 8 show that the therapeutic effect of IL-17, IFNy and TNF[alpha] pretreated MSCs on CIH is dependent on the expression of iNOS.

Figure 9 includes Figures 9A, 9B, 9C, 9D, 9E, and 9F showing that IL-17 can reverse the inhibition of gene expression by the RNA binding protein AUF1.

Panels A-D of Figure 10 show that IL-17 enhances the stability of iNOS mRNA by modulating the level of AUF1.

Panels A-B of Figure 11 show that AUF1 is a key molecule that mediates IL-17 signaling.

Panels A-D of Figure 12 show that AUF1 is a key molecule that mediates IL-17 enhancing MSC treatment of CIH.

detailed description

Through extensive and intensive research, the present inventors have unexpectedly discovered that IL-17 can significantly enhance the immunosuppressive function of mesenchymal stem cells and inhibit the proliferation of T cells; this function enhances the mRNA of iNOS by decreasing the expression level of the RNA binding protein AUF1. The stability is achieved by up-regulating the expression of iNOS in MSC. In vivo and in vitro experiments demonstrated that IL-17 pretreated MSC can effectively treat CONA-induced liver injury. The present invention has been completed on this basis.

the term

The term "treating" refers to the administration of interleukin-17 of the present invention to a subject in need of treatment for the purpose of curing, alleviating, ameliorating, alleviating, affecting the disease, symptoms, and disease predisposition of the subject.

The term "therapeutic subject" refers to rats, humans, and other mammals.

The term "therapeutically effective amount" refers to an amount of interleukin-17 that is capable of achieving a therapeutic purpose in a subject. It will be understood by one of ordinary skill in the art that the "therapeutically effective amount" may vary depending on the route of administration of interleukin-17, the pharmaceutical excipients used, and the combination with other drugs.

Mesenchymal stem cells (MSC)

The inventors' research results show that MSC exerts an immunosuppressive function on T cells through the combined action of nitric oxide (NO) and chemokines. T cells secrete a large number of inflammatory cytokines, including IFNγ, TNFα, IL-1α and IL-1β, after activation of their receptor (TCR). These inflammatory cytokines stimulate MSCs to secrete large amounts of iNOS and chemokines. Chemokines are capable of recruiting T cells to the periphery of MSCs and inhibiting T cell proliferation through the metabolite NO of iNOS. Furthermore, the inventors have also found that the transcription factors C/EBPβ and STAT1 are critical in the induction of iNOS production. The present inventors also studied different mechanisms by which MSCs of different species are mediated in immunosuppression of T cells. For mice, iNOS is a key molecule for MSCs to suppress T cells, while for humans, IDO is a key molecule for MSCs to inhibit T cells. However, other researchers believe that HLA-G5, TGF-β and IL-10 are key molecules that mediate the immunosuppression of human MSCs.

Immunosuppressive factor

As used herein, "immunosuppressive factor" refers to a factor or molecule that can downregulate the body's excessive immune response. Excessive immune response, including B cells, T cells, natural killer cells, macrophages, dendritic cells, neutrophils and other cell-mediated immune responses beyond the normal range of the body, may lead to inflammatory diseases, such as allergic reactions , autoimmune diseases, etc.

Preferred immunosuppressive factors in the present invention include iNOS, indole-2,3-dioxygenase, PGE2, TSG6, IL-6, HO-1, IL-10, PD-L1, Galetin and/or B7- H4.

Interleukin-17 or its derivative and preparation method thereof

As used herein, "interleukin-17" or "IL-17" refers to a protein which has (a) and Zhengbin Yao et al. Human IL-17: a novel cytokine derived from T cells. J. Immunol. 155(12), 5483-5486 (1995); and the basic amino acid sequence of human/mouse interleukin-17 described in Submitted (11-DEC-1995) Jacqueline Kennedy, Immunology, DNAX Research Institute and (b) with natural Interleukin-17 has the same biological activity. The interleukin-17 of the present invention includes, but is not limited to, human interleukin-17, recombinant human interleukin, murine interleukin-17, and/or recombinant murine interleukin-17. In certain embodiments, the amino acid sequence of interleukin-17 is:

Human IL-17 amino acid sequence: (155aa)

1 MTPGKTSLVS LLLLLSLEAI VKAGITIPRN PGCPNSEDKN FPRTVMVNLN IHNRNTNTNP

61 KRSSDYYNRS TSPWNLHRNE DPERYPSVIW EAKCRHLGCI NADGNVDYHM NSVPIQQEIL

121 VLRREPPHCP NSFRLEKILV SVGCTCVTPI VHHVA (SEQ ID NO.: 1)

Mouse IL-17 amino acid sequence: (158aa)

1 MSPGRASSVS LMLLLLLSLA ATVKAAAIIP QSSACPNTEA KDFLQNVKVN LKVFNSLGAK

61 VSSRRPSDYL NRSTSPWTLH RNEDPDRYPS VIWEAQCRHQ RCVNAEGKLD HHMNSVLIQQ

121 EILVLKREPE SCPFTFRVEK MLVGVGCTCV ASIVRQAA (SEQ ID NO.: 2)

Activation of the IL-17 signaling pathway activates the expression of many pro-inflammatory genes, similar to the activation of some classical innate immune receptors, including IL-1R (IL-1 receptor) and TLR (Toll-like receptor). Wait. Similar to IL-1R and TLR, IL-17RA binds to IL-17 and interacts with the adaptor proteins Act1 and TRAF6 to activate NFκB signaling pathway and promote target gene expression. IL-17 activates the NFκB pathway and activates the MAPK signaling pathway. Activation of MAPK leads to activation of AP1 and promotes transcription of target genes. In addition, MAPK activation can also promote gene expression by enhancing mRNA stability. For IL-17, enhancing mRNA stability is an important part of its promotion of gene expression. Among them, Act1 is an important molecule that mediates IL-17 to enhance mRNA stability.

In the inflammatory microenvironment, IL-17 is mainly secreted by Th17 cells, and can also be produced by other cells, including γδT cells, NKT cells, NK cells, neutrophils and eosinophils. As a pro-inflammatory factor, IL-17 is involved in the pathogenesis of many autoimmune diseases. In the present invention, the inventors have found that IL-17 can enhance the immunosuppressive function of MSC in the presence of MSC, thereby effectively inhibiting T cell proliferation, indicating that IL-17 can inhibit the immune response in the presence of MSC.

The term "substantially identical amino acid sequence" refers to a difference in sequence or caused by one or more amino acid changes (deletion, addition, substitution), but such alteration does not substantially reduce its biological activity, ie, by binding to IL- 17 target cell receptors and biological functions. Any interleukin-17 that meets the "substantially identical" requirement is included in the invention, whether it is glycosylated (ie derived from natural or derived from eukaryotic expression systems) or non-glycosylated (ie source) In prokaryotic expression systems or chemically synthesized).

"Interleukin-17" also includes PEGylated IL-17 and covalently modified IL-17 protein. For example, various activated polyethylene glycols (PEG) having a molecular weight of 5,000 to 100,000 can be used to polymerize IL-17 to prolong its half-life. For specific procedures, see Greenwald et al., Bioorg. Med. Chem. Lett. 1994, 4, 2465; Calettiti et al., IL Farmaco, 1993, 48, 919; Zalipsky and Lee, Polyethylene glycol chemistry: biology Technology and Biomedical Applications, edited by J. M. Harris, Plenum Press, N.Y., 1992.

The interleukin-17 of the present invention can be cloned and expressed by genetic recombination techniques. Host cells for expression include prokaryotic cells, yeast cells, or higher eukaryotic cells. In addition to prokaryotic cells, eukaryotic cells such as filamentous fungi or yeast are equally suitable for expressing or cloning the interleukin-17 of the present invention. The host cell of the interleukin-17 of the present invention for expressing glycosylation is derived from a multicellular organism. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, plant cells. Examples of suitable mammalian host cells include Chinese hamster ovary cells (CHO), COS cells. One of ordinary skill in the art will know how to select a suitable host cell.

The above host cells are transfected or transformed with an interleukin-17 expression vector or a cloning vector, and can be cultured in a conventional nutrient medium, which is modified to be suitable for inducing a promoter and a selective transformant. (selecting transformant) or amplifying the interleukin-17 encoding gene sequence. Selection of culture conditions such as medium, temperature, pH, etc. will be known to those of ordinary skill in the art. General principles, protocols, and techniques for how to maximize cell culture fertility can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

The interleukin-17 of the present invention can be directly expressed not only by genetic recombination, but also by forming a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence located at the N-terminus of the mature protein or polypeptide, or may be located in a mature protein or polypeptide. N-terminal other polypeptide fragments with specific cleavage sites.

IL-17 dimer and preparation method thereof

The DNA sequence encoding the IL-17 dimer or fusion protein of the present invention can be all synthetically synthesized. The coding DNA sequences of the IL-17 first monomer and/or the IL-17 second monomer can also be obtained by PCR amplification or synthesis and then spliced together to form a DNA sequence encoding the fusion protein of the present invention.

In order to increase the expression level of the host cell, the IL-17 dimer coding sequence can be engineered, for example, using host cell-preferred codons to eliminate sequences that are detrimental to gene transcription and translation. In the present invention, the yeast cell or mammalian cell-preferred codon can be used, and the IL-17 dimer gene can be detected by computer DNA software, and the sequence which is not conducive to gene transcription and translation in the gene, including the inclusion, is excluded. Sub-shearing site, transcription termination sequence, and the like.

After obtaining the DNA sequence encoding the novel fusion protein of the present invention, it is ligated into a suitable expression vector and transferred to a suitable host cell. Finally, the transformed host cells are cultured, and the novel fusion protein of the present invention is obtained by isolation and purification.

The term "vector," as used herein, includes plasmids, cosmids, expression vectors, cloning vectors, viral vectors, and the like.

In the present invention, various carriers known in the art such as commercially available carriers can be used. For example, a commercially available vector is selected, and then a nucleotide sequence encoding a novel fusion protein of the present invention is operably linked to an expression control sequence to form a protein expression vector.

As used herein, "operably linked" refers to a condition in which portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if a signal peptide DNA is expressed as a precursor and is involved in the secretion of a polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence. Generally, "operably linked to" means adjacent, and for secretory leader sequences means adjacent in the reading frame.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

After obtaining the transformed host cell, the cell can be cultured under conditions suitable for expression of the fusion protein of the present invention to express the fusion protein. The expressed fusion protein is then isolated.

Pharmaceutical composition and method of administration

The pharmaceutical compositions of the present invention comprise a safe or effective amount of the IL-17 or derivative thereof of the present invention and a pharmaceutically acceptable excipient or carrier. By "safe, effective amount" it is meant that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. In general, the pharmaceutical composition contains 0.001 to 1000 mg of IL-17 or a derivative/agent thereof, preferably 0.05 to 300 mg of IL-17 or a derivative/agent thereof, more preferably 0.5 to 200 mg of IL-17 or Its derivatives/agents.

The IL-17 or a derivative thereof of the present invention and a pharmacologically acceptable salt thereof can be formulated into various preparations comprising the IL-17 or derivative thereof of the present invention or a pharmacologically acceptable amount thereof in a safe and effective amount Salts and pharmaceutically acceptable excipients or carriers. By "safe, effective amount" it is meant that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the compound is determined according to the specific conditions such as the age, condition, and course of treatment of the subject.

"Pharmacologically acceptable excipient or carrier" means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity . By "compatibility" it is meant herein that the components of the composition are capable of intermixing with the compounds of the invention and with each other without significantly reducing the potency of the compound. Examples of pharmaceutically acceptable excipients or carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants ( Such as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as

), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.

When IL-17 or a derivative thereof of the present invention is administered, it can be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously) or topically.

The IL-17 or derivative thereof of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

The microcapsule containing the interleukin-17 of the present invention or a derivative thereof can be used for the sustained release administration of the interleukin-17 of the present invention. The microcapsule sustained release drug delivery technology of recombinant protein has been successfully applied to recombinant human growth hormone (rhGH), recombinant human interferon (rhIFN), interleukin-2 and MNrgp120 (Johnson et al., Nat. Med., 2:795- 799 (1996); Yasuda, Biomed. Ther 27: 1221-1223 (1993); WO 97/03692, WO 96/40072, WO 96/07399; US Pat. No. 5,654,010.

The sustained release preparation of the interleukin-17 of the present invention or a derivative thereof can be produced by a lactic acid glycolic acid high polymer (PLGA) having good biocompatibility and broad biodegradability. The degradation products of PLGA, lactic acid and glycolic acid can be quickly eliminated by the human body. Moreover, the degradation ability of the polymer can be extended from several months to several years depending on its molecular weight and composition (Lewis, "Controlled release of bioactive agents form lactide/glycolide polymer," in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41)).

When a pharmaceutical composition is used, a safe and effective amount of IL-17 or a dimer thereof of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective effective dose for 60 kg. For a person weighing, the dose is usually from 0.01 to 300 mg, preferably from 0.5 to 100 mg. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

The main advantages of the invention are:

(1) It was first revealed that IL-17 can significantly enhance the immunosuppressive function of mesenchymal stem cells.

(2) It was first discovered that IL-17 has the ability to inhibit the proliferation of T cells.

(3) It was demonstrated for the first time that IL-17 up-regulates the expression of iNOS in MSC by decreasing the expression level of the RNA binding protein AUF1 to enhance the mRNA stability of iNOS, thereby achieving immunosuppression of mesenchymal stem cells.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples that do not specify the specific conditions are usually The conditions are as described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Materials and Method

Experimental Materials

The reagents and experimental materials used in the present invention are commercially available, and the sources of the biological materials are as follows.

Recombinant mouse IFNγ, TNFα, IL-17A, anti-IL-17A antibody was purchased from eBiosciences (La Jolla, CA); recombinant mouse IL-2 was purchased from R&D Systems (Minneapolis, MN); anti-β-actin, GAPDH, iNOS , p65, p-IκBα, p-p65, p-JNK, p-ERK1/2 antibody were purchased from Cell Signaling Technology (Danvers, MA); anti-Act1 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX); L-NMMA, Propidium iodide, actinomycin D was purchased from Sigma-Aldrich (St. Louis, MO); Concanavaline A (ConA) was purchased from Vector Labs (Burlingame, CA).

C57BL/6 mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. The animals were raised in a clean environment at the Experimental Animal Science Department of the School of Medicine, Shanghai Jiaotong University.

Cell preparation method

(1) Isolation and culture of mesenchymal stem cells:

The experimental mice were sacrificed by cervical dislocation and then immersed in 75% alcohol for 3-5 minutes. The two hind legs of the mice were cut with surgical scissors, and two of the tibia and femur were taken and immersed in DMEM complete medium (containing twice as much penicillin and streptomycin), and the muscle tissue around the bone was removed with surgical scissors. Cut the ends of the removed bones to leave the cavity, see the bone marrow; use a 10 ml syringe to draw 5-10 ml of DMEM medium (without FBS) to the bone cavity and rinse the bone marrow into a 50 ml centrifuge tube. After centrifugation at 350 g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in 5 ml of DMEM complete medium, counted under a microscope, transferred to a T25 or T75 cell culture flask at a seeding density of 5 × 10 ⁶ /cm ² , and placed in a culture. Incubate in a box (37 ° C, 5% CO ₂ ). After 24 hours, the cells were removed by liquid exchange, and the cells were changed every two days. The cells were purified by continuous liquid exchange. Finally, single cell clones were selected by 96-well plate dilution method and amplified. The selected clones were characterized for in vitro differentiation (osteogenesis and adipogenesis) and surface markers, and the mesenchymal stem cells used in all experiments were used before 20 generations. All mesenchymal stem cells were cultured using DMEM complete medium.

(2) Preparation of T cell blasts

Mouse mononuclear cell suspension was counted and cultured in a 10 cm culture dish at a density of 1×10 ⁶ cells/ml. Anti-mouse CD3 antibody (1 μg/ml) and anti-mouse CD28 antibody were added to the 1640 complete medium. (1 μg/ml). After 48 hours, the cells were collected and centrifuged for 500 g for 5 minutes, and the supernatant was collected as Sup-CD3 (small mouse spleen cell culture supernatant stimulated with anti-CD3 and anti-CD28 antibody for 48 hours) while collecting the cell pellet. The cell pellet was resuspended in 1640 complete medium, counted and cultured in a 10 cm culture dish at a density of 1 × 10 ⁶ cells/ml (without anti-CD3 and anti-CD28 antibodies), and IL-2 (200 U/ml was added to the culture solution). After 48 hours of incubation, the cells grow into a mass, which is T cell blasts.

T cell proliferation assay (3H-Tdr incorporation method)

3H-Tdr (0.5 μCi per well) was added to a 96-well plate for detecting T cell proliferation, and cultivation was continued in an incubator, and after 6 hours, the 96-well plate was placed in a -80 °C refrigerator. Until the day of the test, the plate was taken out and placed in an oven at 37 ° C to transfer all the isotope taken up by the cells to the glass cellulose membrane. The membrane was dried using a microwave oven and then added to the scintillation fluid. The cpm value was read on a Wallac MicroBeta liquid scintillation counter to measure the amount of 3H incorporated.

Apoptosis detection

After collecting the cells, wash them with PBS, and add Propidum Iodide staining buffer (PBS containing 0.2% saponin, 50 μg/ml propidium iodide, freshly added RNase A 10 μg/ml) 400 to each sample. The μl was resuspended, stained at room temperature for 30 min, and the DNA content was detected by flow cytometry. .

RNA extraction and gene expression analysis

RNA was extracted by following the instructions of the TIANGEN Cell RNA Extraction Kit. Refer to the TIANGEN TIANScript cDNA First Strand Generation Kit for inversion of RNA to cDNA. Fluorescence quantitative PCR was performed using TAKARA fluorescent quantitative PCR reagent with reference to its instructions. Using β-actin as an internal reference, the results were analyzed by 2-△△CT method, and all gene expression was converted to a multiple of β-actin. The primer sequences used are as follows:

AUF1:

Forward primer 5'-CAGCAGAGTGGTTATGGGAAAGTATCC-3' (SEQ ID NO.: 3);

Reverse primer 5'-GACAGGAGCCACCTTCAAATGAATC-3' (SEQ ID NO.: 4);

--actin:

Forward primer 5'-CCACGAGCGGTTCCGATG-3' (SEQ ID NO.: 5);

Reverse primer 5'-GCCACAGGATTCCATACCCA-3' (SEQ ID NO.: 6);

CCL2:

Forward primer 5'-TCTCTCTTCCTCCACCACCATG-3' (SEQ ID NO.: 7);

Reverse primer 5'-GCGTTAACTGCATCTGGCTGA-3' (SEQ ID NO.: 8);

CCL5:

Forward primer 5'-TTTCTACACCAGCAGCAAGTGC-3' (SEQ ID NO.: 9);

Reverse primer 5'-CCTTCGTGTGACAAACACGAC-3' (SEQ ID NO.: 10);

CXCL9:

Forward primer 5'-AGTGTGGAGTTCGAGGAACCCT-3' (SEQ ID NO.: 11);

Reverse primer 5'-TGCAGGAGCATCGTGCATT-3' (SEQ ID NO.: 12);

CXCL10:

Forward primer 5'-TAGCTCAGGCTCGTCAGTTCT-3' (SEQ ID NO.: 13);

Reverse primer 5'-GATGGTGGTTAAGTTCGTGCT-3' (SEQ ID NO.: 14);

IL-6:

Forward primer 5'-GAGGATACCACTCCCAACAGACC-3' (SEQ ID NO.: 15);

Reverse primer 5'-AAGTGCATCATCGTTGTTCATACA-3' (SEQ ID NO.: 16);

IL-17RA:

Forward primer 5'-AGTGTTTCCTCTACCCAGCAC-3' (SEQ ID NO.: 17);

Reverse primer 5'-GAAAACCGCCACCGCTTAC-3' (SEQ ID NO.: 18);

IL-17RC:

Forward primer 5'-GCTGCCTGATGGTGACAATGT-3' (SEQ ID NO.: 19);

Reverse primer 5'-TGGACGCAGGTACAGTAAGAAG-3' (SEQ ID NO.: 20);

iNOS:

Forward primer 5'-CAGCTGGGCTGTACAAACCTT-3' (SEQ ID NO.: 21);

Reverse primer 5'-CATTGGAAGTGAAGCGTTTCG-3' (SEQ ID NO.: 22).

mRNA half-life determination

On the first day, mesenchymal stem cells were passaged to 5 6-well plates: 1.5 × 10 ⁵ cells/well, 1.6 ml of complete medium was added to each well, and the cells were placed overnight to allow the cells to adhere.

The next day, at 12 noon, IFNγ+TNFα was added to the 6-well plate, and IFNγ+TNFα+IL-17 cytokine was stimulated to a final concentration of 10 ng/ml. After 6 hr, Act.D (5 μg/ml) was added to the 6-well plate at 18 o'clock in the evening, and RNA was collected at 0, 0.5 hr, 1 hr, 2 hr, 3 hr, and 4 hr for 5 time points, each time point corresponding to each treatment. There are three duplicate wells, and the lysate is added and mixed, and then placed at -80 °C.

On the third day, total RNA was extracted and the RNA concentration was determined.

On the fourth day, RNA was reverse transcribed into cDNA for real-time quantitative PCR.

On the fifth day, the results were analyzed by 2-△△CT method with β-actin or GAPDH as internal parameters. The amount of RNA per repeat at each time point was expressed as ratio to β-actin/GAPDH, with average RNA at time 0 Using the amount as a reference, the percentage of the amount of repeating-well RNA in each of the remaining time points relative to the zero-point reference value was calculated. After the EXCEL calculation was completed, Graphpad Prism 5 software was used for the semi-logarithmic curve of mRNA decay, wherein the Y-axis is the percentage and expressed in logarithm; the X-axis is the time point and is linearly represented. The half-life of mRNA was calculated according to the slope k value of the nonlinear regression curve fitted by the software, and the formula was calculated as t1/2=ln(2/k).

RNA interference

Plasmid transfection was performed by electroporation using the kit Amaxa Cell Line Nucleofector Kit V, with reference to its instructions, as follows.

Plasmid: shCTRL, shAUF1, shAct1, GFP plasmid (included in the kit).

Four 6-well plates were taken out, and 4 wells of each of the 6-well plates were added to 1 ml of DMEM complete medium, and pre-equilibrated in a 37 ° C incubator.

The Nucleofector Solution and Supplement in the kit were taken out, equilibrated to room temperature, and the Solution was prepared according to Nucleofector Solution: Supplement = 4.5:1, 100 μl per reaction.

Two 10 cm dish mouse mesenchymal stem cells were digested, collected, washed once with PBS, and then counted.

Pipette 1.5×10 ⁶ cells into 4 clean sterile 1.5 ml centrifuge tubes, centrifuge at 90 g for 10 min at room temperature, and remove the supernatant as much as possible.

The cells were resuspended in 100 μl of Solution, and 2 μg of the corresponding plasmid was added to each centrifuge tube, which was gently blown.

Transfer the above solution to the electric rotor without air bubbles and cover.

Turn on the electro-rotator, select the U-023 program, place the electric rotor into the holder of the electro-rotator, and press the X button to start the electro-rotation procedure.

Immediately after the program was run, remove the electric rotor and quickly add 500 μl of pre-equilibrated DMEM complete medium to the cup. After homogenizing with a pipette, the cells were transferred to 4 wells of a 6-well plate on average for culture.

On the next day, the well plate was taken out, and the cells transfected with the GFP plasmid were observed by fluorescence microscopy. It can be seen that more than 80% of the cells emitted green fluorescence, that is, the transfection efficiency was over 80%. All 6-well plates were exchanged.

(10) On the third day, positive cells were screened with 250 μg/ml of hygromycin B.

(11) After 2-3 weeks of screening, uniform positive cells were obtained, and a small amount of cell lysed protein was taken out, and Western Blot was used to identify the effect of knockdown of the target protein. If the effect is good, the stable cell line is established successfully, and the next experiment can be carried out.

Two cells, AUF1 knockdown and Act1knockdown, were established according to this method.

Western blot

The cells can be collected by cell scraping or trypsinization. After washing with PBS, the collected cells are added with a certain amount of RIPA lysate, mixed by blowing, and placed on ice for 30 minutes, shaking for 3-4 times; Transfer at high speed 13,000 rpm for 30 min at low temperature; transfer the supernatant to a new centrifuge tube and place on ice for later use. Protein concentration was determined using Bio-Rad's Protein Assay Dye Reagent Concentrate (Bradford method). SDS-polyacrylamide gel was prepared by using 8% separating glue and 5% laminated glue. The sample was added to 5× loading buffer by volume ratio, and boiled at 100 ° C for 10 minutes, then cooled at room temperature and loaded. Each sample contains about 50-100μg of protein; each gel contains a protein marker; the electrode is inserted and electrophoresis is started, first 80V electrophoresis for 30min, then 120V electrophoresis for 60min. After the gel is removed, the laminated gel is removed and placed in an electrotransfer buffer; the NC membrane (nitrocellulose membrane) is placed in a transfer buffer for about 10 minutes; from bottom to top, the sponge, filter paper, and membrane are placed. , gel, filter paper, sponge are placed in turn, each layer should pay attention to discharge air bubbles, connect the transfer device, and then turn on the power, constant voltage 100V, 2 hours; after the transfer is finished, turn off the power, remove the membrane, according to the protein Marker and molecular weight of the target protein cut out the strip at the corresponding position and mark it. Place the film in a plastic box, add an appropriate amount of blocking solution, and leave it on the shaker for 2 hours at room temperature; wash 3 times with 1×TBST solution for 3 minutes each time; then transfer the film into a plastic bag and add it to the diluted solution. The first antibody was shaken overnight at 4 °C. The film was taken out the next day and then washed 3 times with 1 x TBST for 10 minutes each time. Add the diluted HRP-labeled secondary antibody and incubate for 1-2 hours at room temperature. Remove the membrane and wash it with 1×TBST for 3 times for 10 minutes each time. Transfer the membrane to the holder and add ECL coloring solution in the dark room. The photoreaction is terminated in a timely manner, and the film is exposed and punched. The content of the target protein is judged based on the thickness and shade of the strip at the corresponding position of the film. Scan the film and save it.

Immunoprecipitation

The cells were collected by trypsinization. After washing with PBS, the collected cells were added with a certain amount of RIPA lysate, mixed by blowing, and placed on ice for 30 minutes, shaking for 3-4 times; then high speed 13,000 rpm Centrifuge at low temperature for 30 min; transfer the supernatant to a new centrifuge tube and let it stand on ice. Protein concentration was determined using Bio-Rad's Protein Assay Dye Reagent Concentrate (Bradford method). The above protein cleavage supernatant was incubated with protein G sepharose beads to remove non-specific binding, and then the supernatant was taken after high-speed centrifugation. 1 μg of Act1 antibody or 1 μg of non-specific IgG control was added to the supernatant, and then 20 μl of protein G sepharose beads was added. Incubate (GE Healthcare) overnight at 4 °C. The beads were washed 4 times with RIPA buffer, then resuspended in 20 μl of 2×SDS loading buffer, boiled at 100 ° C for 10 min, and then protein expression was detected by Western Blot.

Establishment of CIH model, cell therapy and evaluation

(1) Mesenchymal stem cells were pretreated with cytokines for 12-16 hours in advance: WT MSC, iNOS-/-MSC or auf1-/-MSC administered IFNγ+TNFα/IFNγ+TNFα+IL-17 (10 ng/ml) cells Factor stimulation.

(2) According to the body weight, mice were intravenously administered with 15 mg/kg of ConA.

(3) After 30 minutes of ConA injection, intravenous injection of mesenchymal stem cells was started, and each mouse was treated with 5 × 10 ⁵ cells. There were 5 mice per treatment group.

(4) Eight hours after ConA injection, the mice were sacrificed, serum was collected, serum ALT levels were measured, and one leaf liver was immersed in 10% formalin for pathological section H&E staining; the remaining livers were all used to isolate mononuclear cells.

(5) Detection of ALT in mouse serum: Refer to the instructions of the ALT test kit produced by Shanghai Yihua Company for operation.

Localization of MSC in injured liver tissue

GFP-MSC was pretreated with cytokine IFNγ+TNFα/IFNγ+TNFα+IL-17 (10ng/ml) for 12-16 hours in advance; 30 minutes after ConA injection, each group received GFP-MSC intravenous injection, each treatment. Mice were treated with 5 x 10 ⁵ cells. Mice were sacrificed 8 hours after ConA injection.

(1) Immediately after the mice were sacrificed, a small leaf liver was directly placed in the OCT and frozen at -80 °C.

(2) The slicer was sliced to a thickness of 10 μm.

(3) The sections were fixed in acetone pre-cooled at -20 ° C for 10 min, dried and immersed in PBS for 20 min.

(4) Wash twice with PBS and add PBS + 3% BSA for 1 hour at room temperature.

(5) Aspirate excess liquid, add primary antibody (anti-GFP diluted 1:400), and wet the box at 37 °C for 2 hours.

(6) Wash PBS three times for 5 min each time, add secondary antibody (Alexa Fluor 594 Donkey anti-Rabbit antibody 1:500) + DAPI (1:500), and wet the box at 37 °C for 2 hours.

(7) Wash the PBS three times, each time for 5 min, dry it, seal it, observe it with a fluorescence microscope and take a picture.

Immunofluorescence staining of cell surface molecules

The cells were resuspended in 100 μl of FACS buffer for staining at 1 × 10 ⁶ . After addition of fluorescently labeled antibodies (eg PE-labeled anti-mouse IL-17RA antibody, PE-labeled anti-mouse CD3 antibody, PerCP/Cy5.5-labeled anti-mouse CD4 antibody, APC-labeled anti-mouse CD8 antibody, APC-labeled antibody Mouse CD45 antibody, APC-labeled anti-mouse CD25 antibody, FITC-labeled anti-mouse CD3 antibody), incubated for 30 minutes at 4 ° C in the dark. Wash twice with FACS buffer. Finally, the cells were resuspended in 400 μl of FACS buffer and analyzed by FACS Calibur flow cytometry.

Immunofluorescence staining of intracellular cytokines or Foxp3

(1) All cells (about 5-10×10 ⁵ ) were washed once with PBS, and blocked with anti-mouse CD16/CD32 antibody for 10 min, on ice, 30 μl of system per tube.

(2) First stain the cell surface molecules, refer to 11.1.

(3) After washing the FACS buffer once, 100 μl of Fixation/Permeabilization buffer was added to each tube, and fixed at 4 ° C overnight.

(4) 400 × g, centrifuge for 5 min, add 1 × Permeabilization buffer 200 μl, mix.

(5) 400 × g, centrifuge for 5 min, add the corresponding cytokine antibody or Foxp3 antibody (antibody diluted with 1 × Permeabilization buffer), 50 μl of each tube, and stain on ice for 1 hour in the dark.

(6) Directly add 200 μl of 1×Permeabilization buffer, mix well, 400×g, and centrifuge for 5 min.

(7) After discarding the supernatant, add 2 μM of 1×Permeabilization buffer, mix, and centrifuge at 400×g for 5 min.

(8) After discarding the supernatant, add 200 μl of FACS buffer, mix well, 400×g, and centrifuge for 5 min. The cell pellet was resuspended in FACS buffer 200 μl and assayed by flow cytometry.

Data processing and statistical analysis

Three or more replicates were set for each treatment group in the experimental results shown, plotted by Graphpad Prism 5 software, and the data shown is expressed as mean ± standard deviation. Statistical analysis of the data was performed using Student's t test or one-way ANOVA. The significance level was determined as α = 0.05, * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

Example 1 IL-17 can enhance the immunosuppressive function of MSC

Studies have shown that the immunosuppressive ability of mesenchymal stem cells is not intrinsic, but is obtained under the induction of inflammatory cytokines including IFNγ, TNFα, IL-1α and IL-1β. Mesenchymal stem cells secrete a large amount of iNOS and chemokines under the stimulation of inflammatory cytokines IFNγ and TNFα. Chemokines recruit T cells to the periphery of mesenchymal stem cells, and the NO, a metabolite of iNOS, inhibits T cell proliferation. effect. It is well known that IL-17 is a pleiotropic pro-inflammatory cytokine that plays an important role in the pathogenesis of many infectious diseases, inflammatory diseases and autoimmune diseases. The present inventors added inflammatory cytokines IFNγ and TNFα to mesenchymal stem cells cultured in vitro, or added IL-17 thereto, and observed the effects of these cytokines on the immunosuppressive ability of mesenchymal stem cells. The present inventors have found that mesenchymal stem cells can only inhibit the proliferation of T cells in the presence of IFNγ and TNFα, while IL-17 can significantly enhance the immunosuppression of mesenchymal stem cells induced by IFNγ and TNFα. Capacity, this effect is manifested by a significant decrease in T cell proliferation (Fig. 1A).

Previous studies have demonstrated that IL-17 activates downstream signaling pathways by binding to heterodimers composed of IL-17RA and IL-17RC on the cell surface. Since IL-17 can act on mesenchymal stem cells and significantly enhance its immunosuppressive ability, the present inventors examined the expression of IL-17RA and IL-17RC in mesenchymal stem cells. The results showed that mesenchymal stem cells can constitutively express IL-17RA and IL-17RC (Fig. 1B, C).

At different stages of the inflammatory response, the levels of inflammatory cytokines are different, so the inventors investigated whether different doses of IFNy and TNF[alpha] have an effect on the enhanced immunosuppressive function of IL-17. The present inventors have found that IL-17 can significantly enhance the immunosuppressive function of mesenchymal stem cells when the levels of IFNγ and TNFα are only 1-2 ng/ml (Fig. 1D, 4E). Even when the levels of IFNγ and TNFα are high (10-20 ng/ml), IL-17 can improve the immunosuppressive function of mesenchymal stem cells. The present inventors also observed the effect of different doses of IL-17 on the property of enhancing the immunosuppressive ability of mesenchymal stem cells. The results showed that approximately 0.5 ng/ml of IL-17 significantly enhanced the immunosuppressive function of mesenchymal stem cells (Fig. 1F).

To further clarify whether IL-17 is involved in the inhibition of T cell proliferation by MSCs in a T cell-mediated immune response, the inventors co-cultured MSCs with anti-CD3 activated splenocytes and added anti to the co-culture system. -IL-17A antibody. The results showed that MSC can significantly inhibit the proliferation of activated spleen cells, while anti-IL-17A The antibody can largely restore the proliferation of activated splenocytes, reversing the inhibition of MSC (Fig. 1G). When the ratio of MSC to spleen cells is 1:40, the reversal effect of anti-IL-17A is most significant; when the ratio of MSC to spleen cells is 1:20, although the reversal effect of anti-IL-17A is weakened , but still statistically significant. In conclusion, the present inventors have shown that IL-17 can significantly enhance MSC immunity against T cells in T cell-mediated immune responses, particularly when the levels of inflammatory cytokines IFNγ and TNFα present in the immune microenvironment are low. Suppress function.

In order to expand the research of the present inventors, the present inventors also examined the effect of IL-17 on its immunosuppressive function on adipose-derived MSCs, and obtained similar results to bone marrow-derived MSCs (Fig. 1H), namely IL- 17 also enhances the immunosuppressive function of adipose-derived MSCs, suggesting that this function of IL-17 may not depend on the source of MSCs.

Figure 1 shows that IL-17 enhances the immunosuppressive function of MSCs.

(A). MSCs were first stimulated with different combinations of cytokines IFNγ, TNFα and IL-17 (each concentration of 2 ng/ml) for 12 hours, and then co-cultured with T cell blasts (MSC and T cell). The ratio of blasts was 1:20), and after 12 hours, T cell proliferation was measured by ³ H-Tdr incorporation. (B). The expression of IL-17RA and IL-17RC on MSC and Raw264.7 cells (positive control) was detected by real-time fluorescent quantitative PCR. (C). Flow cytometry was used to detect the expression of IL-17RA on MSC surface. (D and E). The MSCs were first stimulated with the cytokine IFNγ+TNFα or IFNγ+TNFα+IL-17 for 12 hours (the concentration of IFNγ and TNFα was diluted in a gradient, the IL-17 concentration was fixed at 10 ng/ml), and then And T cell blasts (requires the addition of IL-2 to proliferate, and does not produce inflammatory cytokines during proliferation). (D) or A1.1 cells (T cell hybridoma cells, can proliferate independently, and do not produce inflammatory cytokines during proliferation, the preparation method of the cells can be referred to the literature (Nature. 1989 Jun22; 339 (6226): 625-6 (E) Co-culture, the ratio of MSC to T cells was 1:20, and after 12 hours, T cell proliferation was measured by ³ H-Tdr incorporation. (F). First, MSC was treated with cytokine IFNγ + TNFα or IFNγ. +TNFα+IL-17 was stimulated for 12 hours (the concentration of IFNγ and TNFα was fixed at 2 ng/ml, and the concentration of IL-17 was diluted), then A1.1 cells were co-cultured, and the ratio of MSC to T cells was 1:10. After 12 hours, T cell proliferation was measured by 3H-Tdr incorporation. (G). MSCs were co-cultured with anti-CD3 and anti-CD28-activated splenocytes at a ratio of 1:40 or 1:20, or Anti-IL-17A was added to the culture medium, and 48 hours later, spleen cell proliferation was measured by ³ H-Tdr incorporation method. (H). First, BMMSC or ADSC was treated with cytokine IFNγ+TNFα or IFNγ+TNFα+IL-. 17 (10 ng/ml for each factor) was stimulated for 12 hours, then co-cultured with A1.1 cells (1:10 ratio of MSC to A1.1), and after 12 hours, ³ H-Tdr T cell proliferation was determined by incorporation assay. Results are expressed as mean Number ± standard deviation, BMMSC: bone marrow-derived MSC; ADSC: fat-derived MSC. The data shown represents the results of three experiments.

Example 2 IL-17 enhances the immunosuppressive properties of MSCs depending on the activity of iNOS

The above results show that IL-17 can significantly enhance the immunosuppressive function of MSCs against T cell proliferation, and the inventors further studied the related mechanisms. Previous studies have suggested that IL-17 can promote the proliferation of human MSCs. Therefore, the inventors first studied whether IL-17 affects the immunosuppressive function by affecting the proliferation of MSC. The present inventors stimulated MSCs with IFNγ+TNFα or IFNγ+TNFα+IL-17 for 24 hours, and then detected the proliferation of MSCs. The present inventors have found that the addition of IL-17 inhibits the proliferation of MSCs while being stimulated by IFNγ and TNFα (Fig. 2A). Therefore, IL-17 does not enhance its immunosuppressive ability by affecting the proliferation of MSCs.

Previous studies by the present inventors have shown that iNOS is an important molecule mediating the immunosuppression of mouse MSCs. The present inventors examined whether this effect of IL-17 enhancing MSC immunosuppression is dependent on the activity of iNOS. The results showed that the effect of IL-17 was completely reversed by the iNOS inhibitor L-NMMA (Fig. 2B), indicating that iNOS plays a crucial role in it.

Figure 2 shows that IL-17 enhances the immunosuppressive properties of MSCs depending on the activity of iNOS.

(A). MSC was treated with cytokine IFNγ+TNFα or IFNγ+TNFα+IL-17 (concentration of IFNγ+TNFα was diluted, IL-17 concentration was fixed at 10 ng/ml) for 24 hours, then 3H-Tdr was used. T cell proliferation was determined by incorporation. (B). First, MSC was stimulated with cytokine IFNγ+TNFα or IFNγ+TNFα+IL-17 (each concentration was 2ng/ml) for 12 hours, and then co-cultured with A1.1 cells (MSC and The ratio of A1.1 was 1:10), or the iNOS inhibitor L-NMMA was added to the medium, and 12 hours later, T cell proliferation was measured by ³ H-Tdr incorporation. All data represent the results of three independent experiments.

Example 3 IL-17 enhances the immunosuppressive function of MSC independent of the effect on T cell apoptosis

The above studies show that IL-17 can synergize with the inflammatory cytokines IFNγ and TNFα to enhance the immunosuppressive function of MSC, and this function is mainly reflected in the inhibition of T cell proliferation and depends on the activity of iNOS (Fig. 1, Fig. 2). . In addition, MSCs can induce lymphocyte apoptosis in the presence of inflammatory cytokines IFNγ and TNFα, a process that is also dependent on NO. In order to determine whether this effect of IL-17 enhancing MSC immunosuppression is dependent on the effect on T cell apoptosis, the inventors pre-stimulated MSC with IFNγ+TNFα or IFNγ+TNFα+IL-17 for 12 hours, and then Co-culture was performed with A1.1 cells at a ratio of 1:10, and A1.1 cells were collected 12 hours later to detect apoptosis. The present inventors have found that the ratio of apoptotic cells in A1.1 cells is less than 0.5% in the case of single culture; and when co-cultured with untreated MSCs, the proportion of apoptotic cells is significantly increased, the ratio is 3-5%. When co-cultured with IFNγ+TNFα pretreated MSC, the proportion of apoptotic cells was further increased, the ratio was 7-10%; A1.1 was co-cultured with IFNγ+TNFα+IL-17 pretreated MSC and Similar to the IFNγ+TNFα pretreated MSC, the proportion of apoptotic cells was still 7-10% (Fig. 3). In addition, this phenomenon does not depend on the concentration of IFNγ and TNFα. Regardless of the concentration of IFNγ and TNFα at 5 ng/ml or 10 ng/ml, IFNγ+TNFα+IL-17 pretreated MSC could not further promote T cell apoptosis. Thus, IL-17 enhances the immunosuppression of MSCs independent of the effects on T cell apoptosis.

The MSC was stimulated with cytokine IFNγ+TNFα or IFNγ+TNFα+IL-17 for 12 hours (the concentration of IFNγ and TNFα was 5 ng/ml or 10 ng/ml, the concentration of IL-17 was always 10 ng/ml), and then The ratio of 1:10 was co-cultured with A1.1 cells. After 12 hours, A1.1 cells were collected, and after PI staining, the DNA content was analyzed by flow cytometry.

Example 4 IL-17 synergistic inflammatory cytokines up-regulate the expression of iNOS in MSC

Previous studies have shown that iNOS and chemokines are the core molecules that mediate MSC immunosuppressive function, and that MSCs that initiate expression of iNOS and some chemokines require induction of the inflammatory cytokines IFNγ and TNFα. The above results of the present inventors show that the property of IL-17 to enhance MSC immunosuppression depends on the activity of iNOS. The present inventors treated MSCs with IFNγ+TNFα or IFNγ+TNFα+IL-17, and then used real-time PCR and Western Blot to determine the expression of iNOS and chemokines. The results showed that IL-17 was able to significantly promote MSC expression of iNOS in combination with IFNγ and TNFα at both mRNA and protein levels (Fig. 4A, B, C, D). At the same time, the present inventors also detected mRNA expression of chemokines involved in immunosuppression of MSC, including CCL2, CCL5, CXCL9, and CXCL10, and found that IL-17 does not cooperate with IFNγ and TNFα to alter the expression of these chemokines. (Fig. 4E). Thus, IL-17 enhances the immunosuppression of MSCs specifically depending on its up-regulation of iNOS. To further verify the up-regulation of IL-17 on some of the gene expression of MSCs in a T cell-mediated immune response, the inventors incubated the activated spleen cell supernatant with anti-IL-17A for a period of time to neutralize the supernatant. IL-17 is then used to stimulate MSCs to detect the expression of some immunoregulatory genes in MSCs. The present inventors have found that activated spleen cell supernatant can significantly induce MSC expression of iNOS, CCL2, CCL5, CXCL9 and CXCL10; neutralizing the supernatant of IL-17 does not induce MSC to express iNOS well (Fig. 4F, G However, expression of the chemokines CCL2, CCL5, CXCL9 and CXCL10 was not affected (Fig. 4G). The above results indicate that IL-17 can promote MSC expression of iNOS in combination with IFNγ and TNFα, but does not affect the expression of chemokines CCL2, CCL5, CXCL9 and CXCL10.

Figure 4 shows that IL-17 synergistic inflammatory cytokines up-regulate the expression of iNOS in MSCs.

(A and E). MSCs were stimulated with different combinations of several cytokines of IFNγ, TNFα and IL-17 (each cytokine concentration was 10 ng/ml), and RNA was extracted from cells after 12 hours, using Real-Time PCR. The method was used to detect the expression of several mRNAs of iNOS, CCL2, CCL5, CXCL9 and CXCL10. (B). MSC with IFNγ+TNFα or IFNγ+TNFα+IL-17 (each cytokine concentration was 10 ng/ml) was treated at different times, and the protein was extracted from the cells, and the protein expression of iNOS was detected by Western Blot. (C). MSCs were stimulated with cytokine IFNγ+TNFα or IFNγ+TNFα+IL-17 for 24 hours (the concentration of IFNγ and TNFα was diluted in a gradient, IL-17 concentration was fixed at 10 ng/ml), and the cell supernatant was collected. The amount of NO was measured using a Greiss reagent. (D). MSCs were stimulated with different combinations of cytokines of IFNγ, TNFα and IL-17 (each cytokine concentration was 10 ng/ml). After 24 hours, the cells were extracted and the protein expression of iNOS was detected by Western Blot. (F). MSCs were stimulated with untreated or anti-IL-17A-treated activated splenocyte supernatant for 24 hours, cell extract proteins were collected, and protein expression of iNOS was detected by Western Blot. (G). MSCs were stimulated with untreated or anti-IL-17A-treated activated splenocyte supernatant for 12 hours, and the cells were harvested for RNA extraction. Real-Time PCR was used to detect iNOS, CCL2, CCL5, CXCL9, and CXCL10. Expression of mRNA.

Example 5 Act1 is a key molecule mediating IL-17 to enhance MSC immunosuppression and related gene expression

Previous studies have demonstrated that IL-17 binds to heterodimers composed of IL-17RA and IL-17RC on the cell surface to activate downstream signaling pathways, while IL-17 binds to IL-17RA and IL-17RC. Afterwards, binding of the adaptor protein Act1 to IL-17R is a central step in activating the downstream signaling pathway of IL-17. In downstream signaling, IL-17 regulates target genes primarily by activating the MAPK and NFκB pathways. It has been reported that IL-17 function and signaling have obvious defects in MEF (mouse embryonic fibroblasts) in which Act1 gene is deleted. The present inventors established Act1knockdown stable cells and corresponding control cells, and compared the phosphorylation of MAPK and NFκB signaling pathway proteins in the two cells after IL-17 stimulation. Similar to Act1 ^-/- MEF, the phosphorylation levels of IκBα, ERK, JNK, and p65 were significantly reduced in Act1knockdown MSCs after stimulation with IL-17, and IL-17 signaling was significantly inhibited (Fig. 5A).

The present inventors also studied the changes in immunosuppressive function and related immunoregulatory gene expression of MSCs stimulated by IFNγ, TNFα and IL-17 after Act1knockdown. The results showed that IL-17 could not effectively increase the level of iNOS expression induced by IFNγ and TNFα after Act1knockdown (Fig. 5B, C), and IL-17 could not enhance the immunosuppressive function of MSC (Fig. 5D). These can be attributed to the inhibition of IL-17 signaling caused by Act1 knockdown, and further confirm the discovery that IL-17 can enhance MSC immunosuppressive function and gene expression.

Figure 5 shows that the role of IL-17 in enhancing enhanced immunosuppression and gene expression in MSC requires the involvement of Act1.

(A). Act1 knockdown cells (shAct1MSC) and corresponding control cells (shCTRL MSC) were treated with IL-17 (10 ng/ml) for a specified time, cells at different time points were collected, and proteins were extracted and detected by Western Blot. Expression of several proteins of p-IκBα, p-ERK, p-JNK and p-p65. P- stands for phosphorylation. (B). shCTRL MSC and shAct1 MSC were treated with IFNγ+TNFα or IFNγ+TNFα+IL-17 (each cytokine concentration was 10 ng/ml) for 24 hours, and the protein was extracted and the protein of iNOS was detected by Western Blot. expression. (C). shCTRL MSC and shAct1 MSC were stimulated with different combinations of cytokines of IFNγ, TNFα and IL-17 (each cytokine concentration was 10 ng/ml). After 12 hours, the cells were extracted and RNA was used, and Real-Time PCR was used. The method detects the expression of iNOS mRNA. (D). First, shCTRL MSC or shAct1MSC was stimulated with cytokine IFNγ+TNFα or IFNγ+TNFα+IL-17 (each concentration was 2 ng/ml) for 12 hours, and then co-cultured with A1.1 cells. (The ratio of MSC to A1.1 was 1:10), and after 12 hours, T cell proliferation was measured by ³ H-Tdr incorporation. All data represent the results of three independent experiments.

Example 6 IL-17, IFNγ and TNFα pretreated MSCs can effectively treat ConA-induced liver injury

Since IL-17 can significantly enhance the immunosuppressive function of MSC induced by IFNγ and TNFα. The present inventors further adopted the ConA-induced hepatitis (CIH) model to explore the role of IL-17 in the regulation of MSC immunosuppressive function in the treatment of diseases. CIH is mainly a T cell-mediated acute hepatitis model, which is a good model for human acute fulminant hepatitis and viral hepatitis. The activation and proliferation of T cells and the destruction of hepatocytes are important pathogenic mechanisms. Many reports suggest that ConA-induced liver damage can be significantly alleviated by drugs that suppress the immune response or remove specific T cell subsets. Surrounding IL-17 can significantly enhance the ability of MSCs to inhibit T cell proliferation. The inventors administered CIH mice to different pretreated GFP-MSCs (MSCs isolated from the bone marrow of GFP transgenic mice) and detected GFP-MSC aggregation in liver tissue approximately 7.5 hours after infusion. Studies have shown that MSC can locate damaged liver sites (Figure 6A). In a further study, the inventors stimulated MSCs with IFNγ+TNFα or IFNγ+TNFα+IL-17 for 12 hours in advance, and infused these MSCs into mice after 30 minutes of ConA injection. The degree of liver damage in the mice was observed and examined. The results showed that IFNγ+TNFα+IL-17 treated MSCs were more effective in treating ConA-induced liver injury than MSC-treated and IFNγ+TNFα-treated MSC-treated groups, mainly in ALT of mouse serum. The content was significantly reduced (Fig. 6B), and the degree of hepatic congestion was significantly reduced (Fig. 6C). So far, the inventors' studies have demonstrated that IL-17 can significantly enhance the immunosuppressive function of MSCs from in vivo and in vitro experiments.

Figure 6 shows that IL-17, IFNγ and TNFα pretreated MSCs are effective in treating ConA-induced liver damage.

(A). After intravenous injection of ConA (15 mg/Kg) for 30 min, GFP-MSC pretreated with control, IFNγ+TNFα or IFNγ+TNFα+IL-17 was administered intravenously (5×10 ⁵ per mouse) cell). After 7.5 hours, a small leaf liver was taken for frozen section, and the localization of GFP-MSC in the liver was detected by immunofluorescence. Blue represents DAPI and red represents GFP. The arrow in the figure points to GFP-MSC with a magnification of 400×. (B, C). After intravenous injection of ConA (15 mg/Kg) for 30 min, MSCs pretreated with different cytokines were intravenously administered (5 mice per group, 5 × 10 ⁵ cells per mouse). After 7.5 hours, blood was taken and the mice were sacrificed for liver testing. (B). Level of serum ALT. (C). Liver observation of mice in each group.

Example 7 The therapeutic effect of IL-17, IFNγ and TNFα pretreated MSCs on CIH depends on their immunosuppressive function on T cells without affecting the proportion of T cell subsets.

MSCs pretreated based on IL-17, IFNγ and TNFα have a good therapeutic effect on ConA-induced liver injury, and the inventors further analyzed the specific mechanism of such pretreatment of MSC to regulate CIH. The present inventors isolated mononuclear cells from the liver tissue of mice, and found that the number of liver mononuclear cells in mice treated with L-17, IFNγ and TNFα pretreated MSCs was significantly reduced (Fig. 7A); Cytological analysis revealed a significant decrease in the number of T cells in CD4 ⁺ and CD8 ⁺ (Fig. 7A). These results indicate that IL-17, IFNγ and TNFα pretreated MSCs have a strong immunosuppressive function in inhibiting T cell proliferation in liver tissue after liver injury, and thus T cell-mediated immune response and damage to liver cells. Both are significantly weakened.

The immunomodulatory function of MSC is not only reflected in its immunosuppression of T cells, but also in the regulation of T cell subset differentiation. In addition, MSC can induce Th17 cells to differentiate into Treg cells, thereby reducing the immune response. To this end, the inventors further investigated whether the therapeutic effects of IL-17, IFNγ, and TNFα-pretreated MSCs observed in the CIH model are also related to the effect of MSCs on T cell subset differentiation. The present inventors isolated mononuclear cells from the livers of mice of different treatment groups, and detected changes in T cell subsets after stimulation with PMA and inomycin in vitro. The results showed that the ratio of CD4 ⁺ T cells to CD8 ⁺ T cells in the liver of normal mice was about 2.3:1 (Fig. 7B). In the liver after ConA induction, the proportion of CD8 ⁺ T cells increased by about one-fold, and the ratio of CD4 ⁺ T cells to CD8 ⁺ T cells was about (0.9 to 1.1): 1; however, after treatment with different treatments, CD4 ⁺ T The ratio of cells to CD8 ⁺ T cells did not change much (Fig. 7B); in CD4 ⁺ T cells, IFNγ ⁺ IL-17A ^- Th1 cells, IFNγ ^- IL-17A ⁺ Th17 cells (Fig. 7C) and Treg cells (Fig. There is no significant difference in the proportion of 7D). Thus, the therapeutic effect of IL-17, IFNγ, and TNFα pretreated MSCs on CIH is dependent on their immunosuppressive function on T cell proliferation without affecting the proportion of T cell subsets.

Figure 7 shows that the therapeutic effect of IL-17, IFNy and TNF[alpha] pretreated MSCs on CIH is dependent on their immunosuppressive function on T cells without affecting the proportion of T cell subsets.

After intravenous injection of ConA (15 mg/kg) for 30 min, mice were intravenously injected with MSCs pretreated with different cytokines (5 mice per group, 5 × 10 ⁵ cells per mouse). Eight hours later, the mice were sacrificed and the liver was taken for follow-up testing. (A). Mononuclear cells were isolated and counted after grinding the liver. The ratio of CD4 ⁺ and CD8 ⁺ T cells was analyzed by flow cytometry and converted to an absolute number. (B). Flow cytometry analysis of the proportion of CD4 ⁺ and CD8 ⁺ T cells in the liver. (C and D). After stimulation of mononuclear cells in the liver with PMA and inomycin for 5 hours, BFA was added to the culture system for further 1 hour, and then IFNγ+IL-17A in liver CD4+ T cells was analyzed by flow cytometry. The ratio of Th1, IFNγ-IL-17A ⁺ Th17, Foxp3 ⁺ Treg cells.

Example 8 The therapeutic effect of IL-17, IFNγ and TNFα pretreated MSCs on CIH depends on the expression of iNOS

Since IL-17 enhances the immunosuppressive effect of MSCs on iNOS activity in vitro (Fig. 2B), the inventors further applied in vivo experiments to verify the central role of iNOS in this process. To demonstrate this, the inventors used iNOS ^-/- MSCs instead of WT MSCs and stimulated cells with a combination of different factors, IFNγ+TNFα or IFNγ+TNFα+IL-17, and then used the different pretreatments of iNOS ^-/- MSCs. To treat CIH mice, observe the efficacy. The results showed that MSCs did not treat ConA-induced liver injury well when MSCs were deficient in iNOS, even if cells were pretreated with IFNγ, TNFα, and IL-17. Mainly, compared with IFNγ+TNFα+IL-17 pretreated WT MSC, both untreated iNOS ^-/- MSC or IFNγ+TNFα+IL-17 pretreated iNOS ^-/- MSC could not effectively inhibit CIH In ALT in murine serum (Fig. 8A), mononuclear cell infiltration in mouse liver was also not reduced (Fig. 8B), and the number of CD4 ⁺ T cells and CD8 ⁺ T cells did not decrease (Fig. 8C), and liver pathological sections were still More necrotic areas were shown (Fig. 8D, 8E). Therefore, the inventors' studies have demonstrated in vivo that iNOS is a key molecule for IL-17 to enhance the immunosuppressive function of MSC.

Figure 8 shows that the therapeutic effect of IL-17, IFNγ and TNFα pretreated MSCs on CIH is dependent on the expression of iNOS.

After intravenous injection of ConA (15 mg/Kg) for 30 min, WT MSCs or iNOS-/-MSCs pretreated with different cytokines were intravenously administered (5 mice per group, 5 × 10 ⁵ cells per mouse) . After 7.5 hours, after taking blood, the mice were sacrificed and the liver was taken for follow-up testing. (A). Detection of serum ALT levels. (B). Mononuclear cells were isolated and counted after grinding the liver. (C). Flow cytometry analysis of the ratio of CD4 ⁺ and CD8 ⁺ T cells and conversion to absolute numbers. (D) Take a small leaf liver for paraffin embedding, H&E staining and photographing, the magnification is 200×. (E). Calculate the percentage of necrotic areas in each group of liver pathological sections in (D). Results are expressed as mean ± standard deviation.

Example 9 IL-17 can reverse the inhibition of gene expression by the RNA binding protein AUF1

Next, the inventors studied the molecular mechanism by which IL-17 synergistic inflammatory cytokines IFNγ and TNFα promote MSC expression of iNOS. The control of the amount of mRNA of many immune molecules produced in an immune response is critical, and once the mRNA is excessively accumulated, it causes excessive activation of immune cells, which in turn triggers a hypersensitivity reaction. In vivo, mRNA accumulation can be regulated by a variety of mechanisms, one of the most important mechanisms is the interaction of mRNA binding protein and mRNA to accelerate the degradation of mRNA. However, in many inflammatory responses, activation of signaling pathways often promotes the expression of some genes, and this promotion needs to be achieved by enhancing the stability of the mRNA. Therefore, controlling the stability of mRNA is critical for the regulation of gene expression in many immune responses. IL-17 is no exception. IL-17 promotes the expression of some inflammatory molecules by increasing the stability of mRNA. Nevertheless, the mechanism of action of IL-17 to improve mRNA stability has not been fully elucidated. The mRNA binding protein plays a crucial role in regulating the stability of mRNA, and it has been reported that the RNA binding protein AUF1 can negatively regulate the stability of iNOS mRNA, and knocking down AUF1 can significantly increase the expression of iNOS mRNA, so the inventors It was investigated whether AUF1, an RNA-binding protein, is involved in IL-17 regulation of iNOS gene expression in MSCs.

The present inventors isolated MSCs from the bone marrow of auf1 ^-/- mice and compared the expression of iNOS in WT MSCs and auf1 ^-/- MSCs after stimulation with IFNγ + TNFα or IFNγ + TNFα + IL-17. The results showed that although IL-17 can up-regulate the expression of iNOS mRNA and protein in WTMSC in combination with IFNγ and TNFα, only IFNγ and TNFα can induce MSC to produce large amounts of iNOS in auf1 ^-/- MSC (Fig. 9A, B). IL-17 does not reflect obvious synergy. This phenomenon was also verified in the AUF1 knockdown MSC. The synergy of IL-17 was also significantly attenuated in the AUF1 knockdown MSC (Fig. 9C, D, E). Not only at the level of gene expression, the inventors found a similar phenomenon in the enhancement of immunosuppression by IL-17: in WTMSC, IL-17 can enhance the immunosuppression induced by IFNγ and TNFα; however, in auf1 ^{- /- In} MSC, only IFNγ and TNFα can induce the strongest immunosuppression without the addition of IL-17 (Fig. 9F). These results suggest that IL-17 can reverse the inhibition of iNOS mRNA expression by AUF1 in WT MSCs; when AUF1 is deleted, iNOS mRNA induced by IFNγ and TNFα loses the inhibitory effect of AUF1 on its expression. At a high level of expression, the promotion of IL-17 is significantly attenuated; AUF1 is likely to be a target for IL-17 to play a role in enhancing gene expression.

Figure 9 shows that IL-17 can reverse the inhibition of gene expression by the RNA binding protein AUF1.

(A). WT MSC or auf1-/-MSCs were stimulated with different combinations of cytokines of IFNγ, TNFα and IL-17 (each cytokine concentration was 10 ng/ml), and RNA was extracted after 12 hours. The expression of iNOS mRNA was detected by Real-Time PCR. (B). WT MSC or auf1-/-MSC was stimulated with IFNγ+TNFα or IFNγ+TNFα+IL-17 (each cytokine concentration was 10 ng/ml) for 24 hours, and the cells were extracted and the protein was detected by Western Blot. Protein expression. (C). The efficiency of knockdown was identified by Real-Time PCR and Western Blot after stable transfection of Act1 shRNA on WT MSC. (D). Actlknockdown cells (shAct1MSC) and corresponding control cells (shCTRL MSC) were stimulated with different combinations of several cytokines of IFNγ, TNFα and IL-17 (each cytokine concentration was 10 ng/ml) for 12 hours. The RNA was extracted from the cells, and the expression of iNOS mRNA was detected by Real-Time PCR. (E). shCTRL MSC or shAUF1 MSC was stimulated with IFNγ+TNFα or IFNγ+TNFα+IL-17 (each cytokine concentration was 10 ng/ml) for 12 or 24 hours, and the cells were extracted and Western blot was used to detect iNOS. Protein. (F). First, WTMSC or auf1-/-MSC was stimulated with cytokine IFNγ+TNFα or IFNγ+TNFα+IL-17 (each concentration was 2ng/ml) for 12 hours, and then it was combined with A1.1. The cells were co-cultured (the ratio of MSC to A1.1 was 1:10), and after 12 hours, T cell proliferation was measured by ³ H-Tdr incorporation. Results are expressed as mean ± standard deviation. The data shown represents the results of four independent experiments.

Example 10 IL-17 enhances the stability of iNOS mRNA by modulating the level of AUF1

The above data indicate that IL-17 can synergize with the inflammatory cytokines IFNγ and TNFα to increase the expression of iNOS in the immunosuppressive function of MSC, but in the absence of AUF1, IL-17 does not reflect this synergistic effect, mainly due to IFNγ. And TNFα can induce the strongest immunosuppression of auf1 ^-/- MSC. These phenomena can be explained by the fact that in WT MSCs, the expression of iNOS requires the induction of IFNγ and TNFα, and these induced iNOS mRNAs rapidly degrade in the presence and interaction of AUF1, and the addition of IL-17 This can reverse the effect of AUF1 on the degradation of mRNA, which promotes the expression of iNOS gene. In the absence of AUF1, the iNOS mRNA induced by IFNγ and TNFα is very stable, and the addition of IL-17 is not shown. Promote the role of gene expression. To test this explanation, the inventors stimulated WT MSCs and auf1 ^−/− MSCs with IFNγ+TNFα or IFNγ+TNFα+IL-17, respectively, to detect the half-life of iNOS mRNA in both cells. It was found that IL-17 significantly enhanced the stability of iNOS mRNA induced by IFNγ+TNFα in WT MSCs, and the half-life of iNOS mRNA was extended from t _1/2 =4.3±1.4 hr to t _1/2 >10 hr (Fig. 10A). In auf1 ^-/- MSC, the stability of iNOS mRNA induced by IFNγ+TNFα was greatly enhanced, and the half-life t _1/2 >10 hr (Fig. 10B), while the addition of IL-17 did not show further prolongation. These results confirm the explanation given by the inventors. For CCL2 and CXCL10 that are not affected by IL-17, IL-17 also does not enhance the stability of these mRNAs accordingly. To further verify these results, the inventors also examined the half-life of iNOS mRNA in AUF1 knockdown MSC, and also obtained similar results as auf1 ^-/- MSC (Fig. 10C).

Since IL-17 can reverse the degradation of iNOS mRNA caused by AUF1, AUF1 is likely to be a key target for IL-17 to play a role. The inventors then studied the molecular mechanism. The present inventors examined changes in AUF1 expression levels of MSCs after stimulation with IFNγ+TNFα or IFNγ+TNFα+IL-17. The results showed that the addition of IL-17 significantly reduced the protein level of AUF1 after stimulation with IFNγ and TNFα (Fig. 10D). Thus, IL-17 enhances the stability of iNOS mRNA by decreasing the level of AUF1, which is a key molecule that enhances immunosuppression and gene expression in IL-17.

Figure 10 shows that IL-17 enhances the stability of iNOS mRNA by modulating the level of AUF1.

(A, B, C). WT MSC (A), auf1-/-MSC (B), shCTRL MSC or shAUF1 MSC (C). IFNγ+TNFα or IFNγ+TNFα+IL-17 (each cytokine) After stimulation for 6 hours at a concentration of 10 ng/ml, Act.D (5 μg/ml) was added to the medium, and the cells were extracted for RNA at a specified time point after the addition of Act.D. The Real-Time PCR method measures the mRNA levels of iNOS, CCL2, and CXCL10 at each time point, and calculates the half-life of mRNA according to the procedures in Materials and Method 8. (D). MSCs were treated with IFNγ+TNFα or IFNγ+TNFα+IL-17 (each cytokine concentration was 10 ng/ml), and the cell extract protein was collected at the specified time point after the addition of cytokines, using Western Blot. Protein expression of AUF1 at each time point was examined.

Example 11 AUF1 is a key molecule mediating IL-17 signaling

In the above study, IL-17 was unable to up ^- regulate the expression of the auf1 ^-/- MSC immunoregulatory gene in combination with the inflammatory cytokines IFNγ and TNFα; the same phenomenon was observed in Act1 ^-/- MSC (Fig. 5). When IL-17 binds to the IL-17 receptor and activates cells, Act1 interacts with the IL-17 receptor, which mediates the downstream signaling, so Act1 is one of the key molecules in the IL-17 signaling pathway. . The inventors first compared the difference in phosphorylation of important signaling molecules in the IL-17 signaling pathway by AUF1 knockdown MSC (shAUF1 MSC) and its normal control cells (shCTRL MSC) after IL-17 stimulation. The present inventors have found that in the case of AUF1 knockdown, phosphorylation of the key molecules p65 (NFκB pathway) and ERK (MAPK pathway) of the IL-17 signaling pathway is significantly attenuated after siRNA is stimulated by IL-17 (Fig. 11A). It is suggested that IL-17 signal transduction is inhibited, and AUF1 may be involved in IL-17 signaling.

In order to further confirm whether AUF1 participates in IL-17 signaling by interacting with Act1, the inventors collected cells at different time points after stimulation with IL-17, and detected the presence of AUF1 and Act1 by co-immunoprecipitation. interaction. The results showed that Act1 had a small amount of binding to AUF1 in the MSC at rest, and the amount of AUF1 bound to Act1 was significantly increased after IL-17 was added (Fig. 11B). Thus, AUF1 does participate in the signal transduction of IL-17 through interaction with Act1.

So far, the present inventors have reached the following conclusions: (1) AUF1 interacts with Act1 during the initiation of IL-17 signal transduction, thereby initiating activation of the downstream NFκB pathway and the MAPK pathway, thereby inducing expression of the target gene. (2) In the presence of IL-17 and other inflammatory cytokines such as IFNγ and TNFα, AUF1 becomes a target of IL-17 regulation, and IL-17 enhances the stability of mRNA of the target gene by decreasing the level of AUF1. Thereby promoting gene expression.

Figure 11 shows that AUF1 is a key molecule that mediates IL-17 signaling.

(A). AUF1 knockdown cells (shAUF1 MSC) and corresponding control cells (shCTRL MSC) were treated with IL-17 (10 ng/ml) for a specified time, cells at different time points were collected, and proteins were extracted, Western Blot method. The expression of p-p65 and p-ERK protein was detected. P- stands for phosphorylation. (B). MSCs were treated with IL-17 (10 ng/ml), and cell extract proteins were collected at the indicated time points after addition of cytokines, and immunologically co-immunized with IgG (control) or anti-mouse Act1 antibody and protein cleavage products. Precipitation, Western Blot detection of Act1 and AUF1.

Example 12 AUF1 is a key molecule mediating the effect of IL-17 on MSC treatment of CIH

Based on AUF1, IL-17 plays a key role in enhancing MSC immunosuppressive function (Fig. 6). Next, the inventors will verify this finding in the CIH model. The present inventors pretreated WT MSC and auf1 ^-/- MSC with IFNγ+TNFα, IFNγ+TNFα+IL-17 for 12 hours, respectively, and then intravenously injected these differently treated cells into CIH mice to observe the therapeutic effect. The present inventors found that IL-17 can significantly enhance the efficacy of WT MSCs for CIH, serum ALT levels, mononuclear cells in the liver, number of CD4 ⁺ T cells and CD8 ⁺ T cells, and degree of liver necrosis, consistent with previous results. Significant decline. IFNγ+TNFα+IL-17 pretreated MSC significantly enhanced the efficacy of WT MSCs on CIH: the mean level of serum ALT decreased from 8000 U/L to 2000 U/L in the untreated group; mononuclear cells in the liver were from the untreated group. 15×10 ⁵ /g liver was reduced to 5×10 ⁵ /g liver; CD4 ⁺ T cells were reduced from 3×10 ⁵ /g liver in the untreated group to 1×10 ⁵ /g liver; CD8 ⁺ T cells were untreated The group's 3.7 × 10 ⁵ /gliver was reduced to 1 × 10 ⁵ /g liver.

However, for auf1 ^-/- MSC, IFNγ + TNFα pretreatment was a good treatment for CIH, and the addition of IL-17 did not enhance the efficacy (Figure 12). So far, the present inventors have demonstrated in vivo that AUF1 is a key molecule that mediates IL-17 enhancing MSC immunosuppressive function and is used for the treatment of CIH.

Figure 12 shows that AUF1 is a key molecule that mediates the effect of IL-17 on MSC treatment of CIH.

After intravenous injection of ConA (15 mg/kg) for 30 min, WT MSCs or auf1 ^-/- MSCs pretreated with different cytokines were intravenously administered (3-5 mice per group, 5 × 10 ⁵ per mouse) cell). After 7.5 hours, blood was taken and the mice were sacrificed for liver testing. (A). Detection of serum ALT levels. (B). Mononuclear cells were isolated and counted after grinding the liver. (C). Flow cytometry analysis of the ratio of CD4 ⁺ and CD8 ⁺ T cells and conversion to absolute numbers. (D) Take a small leaf liver for paraffin embedding, H&E staining and photographing, the magnification is 200×. (E). Calculate the percentage of necrotic areas in each group of liver pathological sections in (D). Results are expressed as mean ± standard deviation.

Example 13 inflammatory factor pretreatment of MSCs can effectively treat cirrhosis

C57BL/6 mice were given peritoneal carbon tetrachloride (CCL ₄ ) weekly for 8 weeks to establish a model of cirrhosis.

Mice treated with different inflammatory factors (10 ng/ml IFNγ+10 ng/ml TNFα or 10 ng/ml IFNγ+10 ng/ml TNFα+10 ng/ml IL-17) were administered to mice at the 8th week of the cirrhosis model. Stem cells (mesensymal stem cells, MSCs) and untreated MSCs were used to observe the therapeutic effects of differently treated MSCs on cirrhosis.

The study found that after cirrhosis was induced in mice, total bilirubin (TB), alanine aminotrans-ferase (ALT), and aspartate amino-transferase (AST) were significantly increased in the serum of mice. Elevated, albumin levels were significantly reduced. Among them, compared with the normal group of mice, the TB level in the cirrhotic model group reached 12-15μmol/L; the ALT level increased to 1200-1800U/L; the AST level increased to 3500-4500U/L; Protein levels are reduced to below 20 g/L.

Infusion of untreated MSCs (1×10 ⁶ ) into cirrhotic model mice can effectively inhibit the levels of TB, ALT and AST in the serum of cirrhotic mice and significantly increase the level of albumin. Seven days after infusion of MSCs, serum TB levels in cirrhotic mice were reduced by 50-60%; ALT levels were reduced by approximately 50%; AST levels were reduced by 60%; and albumin levels were increased by 5%.

After MSCs (1×10 ⁶ ) pretreated with inflammatory factor IFNγ+TNFα for 12 hours, 7 days after infusion into cirrhotic mice, serum TB levels in cirrhotic mice decreased by about 70%; ALT levels decreased. 70-80%; AST levels are reduced by about 70%; albumin levels are increased by 20%. Shows better treatment of cirrhosis. Therefore, inflammatory factors pretreatment of MSCs can effectively improve the therapeutic effect of MSCs on cirrhosis.

IFNγ+TNFα+IL-17 pretreated MSCs (1×10 ⁶ ), and after 7 days of infusion into cirrhotic mice, serum TB levels in cirrhotic mice decreased by 85%; ALT levels decreased by about 80-85%. The AST level was reduced by about 80-90%; the albumin level was increased by 30%. The addition of IL-17 showed a significant synergistic effect and could significantly improve the therapeutic effect of MSCs on cirrhosis.

discuss

The discovery that IL-17 enhances the immunosuppression of MSCs has enabled the present inventors to further study the ability of inflammatory cytokines to regulate MSC immunoregulation, and also laid a foundation for the clinical application of MSCs. In the traditional sense, IL-17 is recognized as an inflammatory factor that promotes immune responses. Many studies have shown that IL-17 is a causative agent of various inflammatory diseases and autoimmune diseases, including rheumatoid arthritis, multiple sclerosis and inflammation. Sexual bowel disease, etc. IL-17 is highly expressed in the serum and tissues of many autoimmune patients, and the symptoms of these autoimmune diseases can be significantly alleviated by using IL-17 neutralizing antibodies or knocking out the IL-17 gene. Nevertheless, IL-17 does not promote an immune response in all pathological conditions. For example, in a DSS (dextran sulfate sodium)-induced mouse enteritis model, the application of IL-17 neutralizing antibodies or knocking out the IL-17 gene may accelerate disease progression. However, the molecular mechanism associated with this phenomenon has not yet been fully elucidated. Studies by the present inventors have shown that IL-17 can exert an immunosuppressive effect in the presence of MSC, and immunosuppression of MSCs when the present inventors neutralize IL-17 in a MSC-T cell co-culture system with a neutralizing antibody. The function will be damaged. These findings by the present inventors provide new ideas and directions for studying the effects of IL-17 under pathophysiological conditions.

ConA-induced liver injury in mice (CIH) is a classic animal model that mimics human viral or autoimmune acute hepatitis. In this model, the acute immune response plays a leading role in mediating liver damage. A variety of immune cells (T lymphocytes, macrophages, NK cells) and immune molecules (cytokines IFNγ, TNFα, IL-10, IL-22, IL-25, etc.) are involved in the pathological process of CIH. Inhibiting an immune response can be a very effective treatment for CIH. In the present paper, the present inventors employed mouse bone marrow-derived MSCs and attempted to apply the IL-17-enhanced MSC immunosuppression property to its treatment of CIH. It was found that untreated MSCs were less effective in treating CIH. This is mainly because the immunosuppressive effect of MSC on T cells is dependent on the stimulation of inflammatory cytokines. Although there are many cytokines including IFNγ and TNFα in the progression of CIH disease, these cytokines can only remain high in a short time. The concentration is not sufficient to confer strong immunosuppressive ability to MSC; however, MSCs pretreated with IFNγ, TNFα and IL-17 can demonstrate the best therapeutic effect, which is consistent with the inventors' conclusions in vitro. Match. The enhancement of IL-17 treatment of CIH by IL-17 depends on the increase of MSC expression of large amount of iNOS after IL-17 addition. For iNOS ^-/- MSC, even if it is pretreated with IFNγ, TNFα and IL-17 in advance, it cannot Make it effective in treating CIH. Therefore, the inventors also verified the discovery that IL-17 enhances immunosuppression in an in vivo experiment.

The present inventors have shown that knockdown of AUF1 can promote the expression of iNOS induced by IFNγ and TNFα in MSC, suggesting the importance of AUF1 for regulation of gene expression in MSC. To further reveal the molecular mechanism by which IL-17 up-regulates MSC gene expression, the inventors examined the effect of IL-17 on the stability of iNOS mRNA, and showed that IL-17 significantly enhanced the stability of iNOS mRNA induced by IFNγ and TNFα. When AUF1 is absent or knocked down, IL-17 does not synergize IFNγ and TNFα to enhance iNOS gene expression, and does not further enhance iNOS mRNA stability. In vitro immunosuppressive experiments showed that auf1 ^-/- MSCs only required the presence of IFNγ and TNFα to exert the strongest immunosuppressive ability and did not require IL-17 supplementation. The present inventors further studied the specific mechanism of AUF1 gene expression in IL-17-enhanced MSCs, and found that MSCs stimulated by IFNγ and TNFα, IL-17 addition can reduce the expression level of AUF1, and this discovery reveals AUF1 involvement. IL-17 regulates the expression of MSC-related genes, that is, IL-17 promotes the stability of iNOS mRNA by decreasing the level of AUF1. The results of co-immunoprecipitation also showed that the interaction between AUF1 and Act1 was significantly enhanced 15 min after IL-17 stimulation. The present inventors also studied the changes in IL-17 signaling pathway protein activation in AUF1 knockdown MSCs, and the results showed that phosphorylation of p65, ERK and the like in the IL-17 signaling pathway was significantly decreased after AUF1 was knocked down, suggesting that AUF1 Knockdown caused impaired IL-17 signal transduction. These data indicate that AUF1 plays a dual role in IL-17-enhanced MSC gene expression: AUF1 is required for IL-17 initiation signaling; whereas when IL-17 synergizes with other cytokines, AUF1 is also IL- The target of 17, IL-17, promotes the stability of related mRNA by decreasing the level of AUF1. Therefore, the present inventors have found for the first time that AUF1 plays an important role in mediating IL-17 enhancing the expression of immunosuppressive genes, and lays a foundation for further elucidating the molecular mechanism of IL-17 action.

In summary, the inventors' research revealed for the first time that IL-17 can enhance the immunosuppressive function of MSC. This effect of IL-17 is mainly achieved by reversing the degradation of mRNA by the RNA binding protein AUF1. The present inventors believe that an in-depth study of IL-17 regulation of MSC immunosuppression will facilitate the better application of MSCs in clinical practice.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Use of a derivative of interleukin-17, interleukin-17 or an agonist thereof, for use in the preparation of a preparation or kit for:

(1) improving the immunosuppressive function of mesenchymal stem cells; and/or

(2) up-regulating the expression of immunosuppressive factors in MSC; and/or

(3) enhancing the stability of mRNA of immunosuppressive factors in MSC; and/or

(4) reducing the expression level of the RNA binding protein AUF1; and/or

(5) inhibiting the proliferation of T cells; and/or

(6) Treatment of hepatitis or liver damage.
The use according to claim 1, wherein the interleukin-17 is a mammalian interleukin-17.
Use of an interleukin-17 antagonist for the preparation of a formulation or kit for:

(1) reducing the immunosuppressive function of mesenchymal stem cells; and/or

(2) down-regulating the expression of immunosuppressive factors in MSC; and/or

(3) reducing the stability of mRNA of immunosuppressive factors in MSC; and/or

(4) enhancing the expression level of the RNA binding protein AUF1; and/or

(5) Promote the proliferation of T cells.
A composition comprising interleukin-17 or a derivative thereof, IFNγ and TNFα.
An isolated MSC cell population characterized in that the MSC cell population consists of or consists essentially of MSC cells, and the MSC cells have enhanced ability to inhibit T cell proliferation,

And, the MSC cells are selected from the group consisting of:

(1) an in vitro pretreated MSC cell population, wherein the pretreatment refers to simultaneous, sequential or sequential treatment with (i) interleukin-17 or a derivative thereof, (ii) IFNγ and (iii) TNFα;

(2) a cell population in which Act1 protein is overexpressed and/or AUF1 protein is deleted or reduced in activity;

(3) an in vitro pretreated MSC cell population, wherein the pretreatment refers to simultaneous, sequential or sequential treatment with IFNγ and TNFα;

(4) A combination of group (1), group (2), and group (3).
A genetically engineered cell strain characterized in that the cell strain is genetically engineered to result in overexpression of an endogenous Act1 protein and/or a decrease in AUF1 protein or activity.
An isolated protein complex characterized in that the protein complex is a protein complex that binds to the Act1 protein and the AUF1 protein.
Use of a protein complex according to claim 7, characterized in that the use is for screening a drug or a compound which promotes or inhibits the formation of the complex by the Act1 protein and the AUF1 protein.
A kit characterized in that the kit contains the following components:

(a) interleukin-17 or a derivative thereof;

(b) IFNγ; and

(c) TNFα;

And instructions for use,

Wherein the components (a), (b) and (c) are respectively located in one or more different containers or in the same container.
A kit comprising the pharmaceutical composition according to claim 4 and instructions for describing the pharmaceutical composition for:

(1) improving the immunosuppressive function of mesenchymal stem cells; and/or

(2) up-regulating the expression of immunosuppressive factors in MSC; and/or

(3) enhancing the stability of mRNA of an immunosuppressive factor; and/or

(4) reducing the expression level of the RNA binding protein AUF1; and/or

(5) inhibiting the proliferation of T cells; and/or

(6) Treatment of hepatitis or liver damage.
A method of treating hepatitis or liver damage, characterized in that the method comprises the steps of:

A therapeutically effective amount of MSC cells is administered to the subject in need thereof.