CN107019793B

CN107019793B - Application of MBL in preparation of medicine for preventing or treating diseases caused by Th17 cells

Info

Publication number: CN107019793B
Application number: CN201710109606.2A
Authority: CN
Inventors: 王凡平; 王明永; 陈晨; 穆永慧; 王帅; 杨帆; 王向鹏; 张晓琴; 郭庆合; 段巨洪
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2017-02-27
Filing date: 2017-02-27
Publication date: 2019-12-24
Anticipated expiration: 2037-02-27
Also published as: CN107019793A

Abstract

The invention discloses an application of MBL in preparation of a medicine for preventing or treating diseases caused by Th17 cells, and belongs to the technical field of new applications of mannan-binding lectin. The technical scheme provided by the invention has the key points that: the application of MBL in the preparation of medicines for preventing or treating diseases caused by Th17 cells, wherein the diseases caused by Th17 cells comprise autoimmune diseases, allergic diseases and infectious diseases. The invention highlights a new function of MBL, not only plays an important role in natural immunity, but also can inhibit the induced differentiation of proinflammatory Th17 cells, and is a new target for treating various autoimmune diseases and inflammatory diseases.

Description

Application of MBL in preparation of medicine for preventing or treating diseases caused by Th17 cells

Technical Field

The invention relates to the technical field of new application of mannan-binding lectin, in particular to application of MBL in preparation of a medicament for preventing or treating diseases caused by Th17 cells.

Background

Mannan-binding lectin (MBL) is an acute phase reaction protein, is mainly secreted by liver cells, is a gelator (collectins) family member in a C-type lectin superfamily, and belongs to Ca⁺A dependent lectin. The human MBL gene is located on chromosome 10q11-q21, MBL peptide chain is composed of 229 amino acid residues, mature MBL has four structural domains, and an N-terminal region rich in cysteine, a collagen-like region, a neck region and a C region capable of carbohydrate recognition are arranged from N end to C end. MBL is a key soluble Pattern Recognition Receptor (PRR) in a natural immune system, can selectively recognize sugar structures on the surfaces of various pathogens (such as bacteria, fungi, viruses and parasites), and plays roles of dissolving and indirectly regulating by activating a complement lectin pathway; or by combining with the surface gelling receptor of phagocyte, the composition can play a direct opsonization role, thereby protecting the body from the invasion of pathogen. Research shows that the concentration of MBL in serum of normal human is about 0.01-10mg/L, and MBL is used as an acute phase reaction proteinIn the acute state, the concentration can be increased by more than 3 times. MBL is called "a key molecule in the innate immune system" and plays an important role in the body's immune system.

Studies have demonstrated that MBL deficiency is the most common immunodeficiency state in humans, mainly due to polymorphisms in the MBL-2 promoter gene and the first exon of the structural gene. MBL deficiency causes the concentration level of MBL in serum to be obviously reduced, and an organism is in an immunodeficiency state and is very easy to infect, and a serious patient can threaten life. Research shows that MBL deficiency is closely related to various infectious diseases, and particularly shows more obvious when the organism is combined with other low immune functions (such as HIV infected patients, tumor patients after drug chemotherapy and children); MBL deficiency is closely associated with a variety of autoimmune diseases, such as MBL deficiency is closely associated with Systemic Lupus Erythematosus (SLE); MBL deficiency may contribute to the deposition of autoimmune complexes, activating the classical complement pathway, leading to joint damage, and thus to the initiation of Rheumatoid Arthritis (RA); MBL is also closely associated with cardiovascular diseases such as atherosclerosis; MBL is also closely related to the development of diabetes.

With the intensive research on MBL, it was found that it plays an important role not only in the third pathway of complement (i.e., mannan-binding lectin pathway), as a key molecule in the innate immune system, but also in the adaptive immune system. In vitro studies have shown that HIV infection with CD4 can be inhibited 100% by MBL pretreatment of HIV-1⁺H9 lymphocytes; the high-concentration MBL (10-50 mg/L) can obviously inhibit the growth of Raji cells of a B lymphoid system; MBL can inhibit TNF-alpha and IL-8 production by HTP1/CD14 cells induced by Candida albicans (C.albicans). Therefore, the deep research on the function of the MBL in the adaptive immune system has important scientific significance and potential medical practice significance.

T lymphocytes are the major players in the adaptive immune response, and CD4 was first discovered⁺T cells are divided into three subpopulations of classical Th1 cells, Th2 cells and regulatory T cells (tregs). Th1 cell mainly secretes IFN-gamma, IL-12 and other cellsFactors control intracellular pathogen (bacterial and viral) infection, Th1 cells are associated with various autoimmune diseases; th2 cell mainly secretes cytokines such as IL-4, IL-5 and IL-13 to control infection of parasite and other extracellular microorganisms, and partially mediate anaphylaxis; treg cells play an important role mainly in maintaining the immune balance of the body. With the continuous and intensive research, the T cells can not completely explain the pathogenesis of some diseases such as autoimmune diseases, infection, anaphylaxis and the like. In recent years, the research finds that the CD4 is independent from Th1 cells, Th2 cells and Treg cells⁺T cells, which are increasingly attracting attention, are called Th17 cells (T helper 17 cells) because they secrete mainly IL-17 cytokines. Th17 cells play a pivotal role in maintaining body balance. It was found that the number of Th17 cells was increased in some murine models of autoimmune disease and in some autoimmune patients, such as Experimental Autoimmune Encephalomyelitis (EAE), collagen-induced arthritis (CIA), Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Multiple Sclerosis (MS), asthma, etc. These findings suggest that intervention in Th17 cells may be a target for the treatment of a variety of autoimmune and some inflammatory diseases.

Recent studies have found that Th17 cells act as a pro-inflammatory CD4 cell⁺T cell subsets play an important role in chronic inflammatory, autoimmune, neoplastic and infectious diseases. Research shows that the nuclear orphan receptor gamma t (the nuclear receptor ROR gamma t) is a key transcription factor for Th17 cell differentiation, the specific transcription factor for promoting human Th17 cell differentiation is RORc, and the transforming growth factor TGF-beta₁（Transforming Growth Factor-β₁) IL-6, IL-21 and IL-23 are important in inducing differentiation of Th17 cells. Therefore, the search for a new factor for inhibiting the Th17 cell differentiation has important scientific significance and clinical medical practice significance for treating various autoimmune diseases. If one wants to reduce the number of Th17 cells, one can block the expression of the specific nuclear transcription factor ROR gamma t and reduce several cytokines inducing the differentiationWhether there are other unknown factors capable of inhibiting the differentiation of Th17 cells is in need of further intensive study.

In recent years, research on related research on MBL immunoregulation has been carried out in this subject group, and the research finds that low-concentration MBL (physiological concentration, 1 mg/L) has a promoting effect on Dendritic Cell (DC) differentiation and maturation and enhances immune response; the high-concentration MBL (super physiological concentration, 10 mg/L) obviously inhibits the growth of B lymphoid line Raji cells; high concentrations of MBL (supraphysiological concentration, 10 mg/L) inhibited TNF- α and IL-8 production by Candida albicans (C.albicans) induced HTP1/CD14 cells. These findings all indicate that MBL plays a different role in immune regulation.

Disclosure of Invention

The invention aims to provide the application of MBL in preparing a medicament for preventing or treating diseases caused by Th17 cells, and mainly discusses the application of MBL to CD4⁺The effect of T cells on inducing differentiation to Th17 cells shows that MBL can directly inhibit CD4⁺The differentiation of the T cells to the Th17 cells is induced, which not only has a theoretical significant breakthrough (the literature report on MBL induced Th17 cells never before), but also has potential clinical medical significance, and provides a new treatment way for treating autoimmune diseases, allergic diseases and infectious diseases caused by the Th17 cells.

The invention adopts the following technical scheme for realizing the aim, and the application of the MBL in preparing the medicine for preventing or treating the diseases caused by the Th17 cells comprises autoimmune diseases, allergic diseases and infectious diseases, wherein the autoimmune diseases comprise systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Graves disease and diabetic nephropathy, the allergic diseases comprise bronchial asthma, allergic purpura and allergic rhinitis, and the infectious diseases comprise chronic hepatitis B virus infection, hepatitis C virus infection, HIV infection and HBV intrauterine infection.

Further preferably, the MBL is obtained by inhibition of CD4⁺The induction of differentiation of T cells to Th17 cells achieves the efficacy of the drug.

Further preferably, the MBL is a purified human plasma native MBL protein by affinity chromatography using a combination of a ligand and a monoclonal antibody.

Further preferably, the concentration of MBL is higher than its physiological concentration in vivo.

More preferably, the concentration of MBL is 10 ng/mL.

The invention has the following advantages:

1. the source of the blood specimen adopted by the invention is umbilical cord blood, and the umbilical cord blood is easier to obtain and relatively more in collected amount compared with peripheral blood, and a large number of hematopoietic stem cells are also contained in the umbilical cord blood, so that the differentiation is easier to induce;

2. the invention obtains a new factor for regulating induced differentiation of Th17 cells by searching, namely MBL directly inhibits CD4⁺Inducing differentiation of T cells to Th17 cells;

3. the flow cytometry detection of the invention is that the transcription factor ROR gamma t antibody specific to Th17 cells is more specific than the detection of IL-17A antibody (IL-17A is mainly secreted by Th17 cells);

4. the invention highlights a new function of MBL, not only plays an important role in natural immunity, but also can inhibit the induced differentiation of proinflammatory Th17 cells, and is a new target for treating various autoimmune diseases and inflammatory diseases.

Drawings

FIG. 1 is immunomagnetic bead sorting CD4⁺A purity profile of T cells;

FIG. 2 is a flow cytometry assay for MBL vs. CD4⁺T cells induced a differentiation pattern towards Th17 cells.

Detailed Description

The present invention is described in further detail below with reference to examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above subject matter of the present invention belong to the scope of the present invention.

Examples

Experimental Material

1. Source of cells for experiments

The umbilical cord blood of the newborn is collected from a third subsidiary hospital of the medical institute of New county and a first people hospital of the New county, the term health newborn in obstetrics and gynecology department of the first people hospital of the New county, and the fetus is immediately taken from the puncture of the umbilical vein of the placenta after delivery and put into a blood-taking bag containing 28mL of preservation solution. All blood samples were sent to the laboratory within 4h after sampling.

2. Primary reagent

Culture medium RPMI1640, Fetal Bovine Serum (FBS) 10 XPBS solution, all from Thermo Fisher Scientific, Inc.

Lymphocyte isolates (Ficoll-Paque PLUS) were Ficoll TMPM400 and a sterile endotoxin (< 0.12 EU/mL) test solution of sodium diatrizoate at a density of 1.077g/mL, available from GE Healthcare Life Sciences.

Anti-human CD3 monoclonal antibody (CD 3 mAb), anti-human CD28 monoclonal antibody (CD 28 mAb), PE-Cyanine7 labeled CD3mAb, FITC labeled CD4mAb, PE labeled ROR γ tmAb, and Cell Stimulation cocktails (plussprotein transport inhibitors) were all purchased from eBioscience, inc; BV605 labeled CD8mAb was purchased from Biolegend.

Human CD4⁺The magnetic bead sorting kit, the MS sorting column and the MACS sorting frame were purchased from Miltenyi Biotec Inc.

Recombinant human MBL was purchased from ACRO Biosystems; human IL-6 and transforming growth factor-beta₁（TGF-β₁) Purchased from Miltenyi Biotec company.

3. Main apparatus and equipment

Clean bench, model SW-CJ-2FD, Suzhou clarification facilities, Inc.

Water bath tank, model SSW-420-2S, Shanghai Bo news corporation.

CO₂Incubator, Thermo company.

Low speed desk centrifuge, model TDL-5, shanghai an xiang scientific instrument factory.

Flow cytometer, FACS Calibur model, BD company, usa.

Inverted microscope, model TS100-F, Nikon, Japan.

Low temperature centrifuge, model Eppendorf 5810R, Eppendorf, germany.

Water purification apparatus, model NW, hel Forece.

4. Preparation of common reagent

(1) 1 XPBS solution

1 part by volume of a 10 XPBS solution +9 parts by volume of distilled water

(2) 1 XPerm Buffer solution

1 part by volume of 10 XPerm Buffer +9 parts by volume of deinized Water

For example: if 10mL of 1 XPerm Buffer solution is needed, 1mL of 10 XPerm Buffer and 9mL of deinized Water are measured and mixed uniformly.

Experimental methods

1. Extraction of human Cord Blood Mononuclear Cells (CBMC)

Human umbilical cord blood mononuclear cells were isolated by density gradient centrifugation according to the lymphocyte isolate (Ficoll) protocol as follows: taking a plurality of 15mL glass tubes, adding 4mL Ficoll into each glass tube, inclining the glass tubes to 45 ℃, slowly adding 8mL of fresh umbilical cord blood diluted by 1:1 on the liquid level of the lymphocyte separation liquid along the tube wall, and keeping the two interfaces clear. Centrifuging at 1500r/min for 30 min. After centrifugation, the liquid level is divided into three layers, the upper layer is a plasma layer, the middle layer is a human lymph separation liquid layer, and the lower layer is mainly a red blood cell layer and a granulocyte layer. The milky cloudy layer between the upper and middle layers is the mononuclear cell layer (CBMC). The cloudy layer was carefully pipetted with a disposable pipette, transferred to a new 15mL centrifuge tube, and the cells washed by adding three volumes of 1 XPBS. Centrifuging at 1500r/min for 10min, discarding the supernatant, and repeating twice. The cell pellet was resuspended in RPMIMedium 1640 medium (10 wt% FBS) and viable cells were counted for use.

2. Human cord blood CD4⁺Magnetic sorting of T cells

Sample preparation

Resuspend the extracted CBMC cells with a buffer, to remove platelets, resuspend the cells with a buffer and centrifuge at 200 Xg for 10-15min, carefully aspirate the supernatant and repeat the wash twice.

Magnetic label CD4⁺T cells

The whole process is completed quickly, and the cells are kept in a precooling state, so that the cap formation phenomenon is prevented.

The incubation temperature is recommended to be 2-8 ℃.

MS column magnetic label is less than or equal to 107 cells.

The cell suspension was treated with a filter with a 30 μm nylon filter mesh for blood, which was wetted with buffer before use.

(1) Counting the cells;

(2) centrifuging the cell suspension at 300 Xg for 10min, and completely discarding the supernatant;

(3) add 80. mu.L of MACS buffer resuspended cells per 107 cells;

(4) add 20. mu.L of CD4 MicroBeads per 107 cells;

(5) mixing and incubating in refrigerator (2-8 deg.C) for 15 min;

(6) (optional step) adding a staining antibody. For example, 10. mu.L of CD4-FITC is added and incubated for 5min in a refrigerator (2-8 ℃) protected from light;

(7) adding 1-2mL of MACS buffer into each 107 cells, centrifuging for 10min at 300 Xg, and completely discarding the supernatant;

(8) adding 500 mul of buffer resuspended cells into each 108 cells, wherein the number of the cells is less than 108, and the volume of the buffer is increased correspondingly when the number of the cells is more than 108;

(9) and (5) carrying out next magnetic sorting.

CD4⁺Magnetic positive sorting of T cells

The appropriate sorting column was selected based on total cell number.

The next step is performed by waiting until the liquid in the separation column is exhausted.

Sorting with MS separation columns

(1) Placing the MS separation column in a MACS classifier;

(2) adding 500 mu L of buffer to rinse the separation column;

(3) 500 μ L of cell suspension was placed in an MS column and the effluent (i.e., unlabeled CD 4) was collected^-T cells);

(4) the MS column was washed 3 times with 500. mu.L buffer and the effluent collected (i.e., unlabeled CD 4)^-T cells);

(5) removing the MS separation column from the MACS classifier, and placing the MS separation column on a proper collection pipe;

(6) add 1mL buffer and place in MS separation column, push plunger immediately to label CD4⁺T cells were placed in a collection tube and repeated twice;

(7) to increase CD4⁺Purity of T cells, CD4 collected in the previous step⁺The T cells may be passed through a second MS separation column, repeating the sorting steps 1-6;

(8) collected CD4⁺T cell suspension, 1500rpm centrifugation for 10min, completely discarding the supernatant, using 10wt% FBS RPMI Medium 1640 Medium heavy suspension cell precipitation, and viable cell count, for use.

3. Coated anti-CD3mAb

1mg/mL of anti-CD3mAb was diluted to 2. mu.g/mL with sterile 1 XPBS and added to 96-well plates at 50. mu.L/well overnight at 4 ℃.

4. Induced differentiation of Th17 cells by different concentrations of MBL

At a rate of 1X 10 per hole⁶Density of individual/mL, sorted CD4⁺T cells were added to 200. mu.L per well of a 96-well cell culture plate pre-coated with 2. mu.g/mL anti-CD3mAb, and anti-CD28mAb (1. mu.g/mL), IL-6 (25 ng/mL) TGF-. beta.was added to each well₁(0.5 ng/mL), different concentrations (1 ng/mL, 5ng/mL, 10 ng/mL) of MBL were added to each experimental group, MBL was not added to the control group, and 3 duplicate wells were provided for each group. The cells were placed at 37 ℃ in 5% by volume CO₂Culturing in an incubator. On day 3 of Cell culture, Cell Stimulation Cocktail (plus protein transport inhibitors 500 ×) was added for 20h Stimulation, and then cells were collected for relevant experiments.

5. Flow cytometry detection of expression of transcription factor ROR gamma t of Th17 cell under different conditions

(1) The cell suspension after each group of culture was collected in 1.5mL EP tubes at 1X 10/tube⁶(ii) individual cells;

(2) centrifuging at 4 deg.C for 5min at 500 Xg, and completely discarding supernatant;

(3) adding 400 μ L of 1 × PBS to resuspend the cell precipitate, centrifuging at 4 deg.C for 5min at 500 × g, discarding the supernatant, and repeating once;

(4) adding 200 μ L of 1 × PBS for resuspension of cells, adding 1 μ L of PEcycline 7-CD3, 2 μ L of FITC-CD4 and 1 μ L of BV605-CD8, respectively, incubating at 4 deg.C in the dark for 30 min;

(5) after incubation, 200. mu.L of 1 XPBS was directly added, at 4 ℃ and 500 Xg, centrifuged for 5min, the supernatant was discarded and repeated once;

(6) adding 200 μ L of 1 XFixation/Permeabilization solution to resuspend the cells, incubating at 4 deg.C for 30min in the absence of light;

(7) after the incubation is finished, 200 mu L of 1 XPerm/Wash Buffer is directly added, the mixture is centrifuged for 5min at 4 ℃ and 500 Xg, and the supernatant is discarded and repeated once;

(8) adding 100 μ L of 1 XPerm/Wash Buffer and 1 μ L of PE-ROR γ t, respectively, incubating at 4 deg.C in dark for 30 min;

(9) after the incubation is finished, 200 mu L of 1 XPerm/Wash Buffer is directly added, the mixture is centrifuged for 5min at 4 ℃ and 500 Xg, and the supernatant is discarded and repeated once;

(10) adding 400. mu.L of 1 XPBS to resuspend the cells, waiting for machine detection, if the machine detection can not be carried out in time, adding 500. mu.L of 4wt% paraformaldehyde to resuspend the cells, and loading the cells for 24-48 h.

Results of the experiment

As can be seen from FIG. 1, high purity CD4 was obtained by immunomagnetic bead sorting⁺A T cell; as can be seen from FIG. 2, MBL inhibits CD4⁺ RORγt⁺Ratio of Th17 cells, it is shown that MBL at high concentrations (10 ng/mL, above physiological concentrations) inhibits CD4⁺The T cells are induced to differentiate to Th17 cells, and can be used as a new target for treating various autoimmune diseases and inflammatory diseases.

The foregoing embodiments illustrate the principles, principal features and advantages of the invention, and it will be understood by those skilled in the art that the invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the invention, and that various changes and modifications may be made therein without departing from the scope of the principles of the invention.

Claims

1. Preparation of mannan-binding lectin for inhibiting CD4⁺Use of a T cell to induce differentiation into a Th17 cell, wherein the mannan-binding lectin is present at a concentration of 10 ng/mL.