CN116355844B

CN116355844B - Establishment and application of SARS-CoV-2 antigen induced cytokine storm model

Info

Publication number: CN116355844B
Application number: CN202310628453.8A
Authority: CN
Inventors: 牛超; 崔久嵬
Original assignee: First Hospital Jinlin University
Current assignee: First Hospital Jinlin University
Priority date: 2023-05-31
Filing date: 2023-05-31
Publication date: 2023-08-18
Anticipated expiration: 2043-05-31
Also published as: CN116355844A

Abstract

The invention discloses a method for establishing and applying SARS-CoV-2 antigen induced cytokine storm model, concretely, the method includes adding SARS-CoV-2 antigen and interleukin into immune cell culture system. The invention provides a model of SARS-CoV-2 antigen induced cytokine storm, which has simple operation and lower requirements on laboratory level and hardware, is suitable for developing effective medicaments for resisting SARS-CoV-2 induced cytokine storm by various scientific research institutions and enterprises, is also suitable for clinically screening effective therapeutic medicaments for SARS-CoV-2 infected patients, and has good application prospect.

Description

Establishment and application of SARS-CoV-2 antigen induced cytokine storm model

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to establishment and application of a SARS-CoV-2 antigen induced cytokine storm model.

Background

Cytokine storm (Cytokine storm), a syndrome of Cytokine release (Cytokine release syndrome, CRS), is an excessive response of the body to infection. When the body is infected by some viruses or the immune function is abnormal, the cytokine balance in the body is broken, the proinflammatory cytokines are continuously and largely produced, and more immune cells are continuously activated to be accumulated at the inflammation part. Excessive immune cells and a variety of pro-inflammatory cytokines can cause tissue congestion, edema, fever, injury, and may also cause other secondary infections and even lead to "systemic inflammatory response syndrome" (sepsis), leading to death in patients due to multiple organ failure.

The main cause of death of moderately severe SARS-CoV-2 infected patients is the occurrence and development of cytokine storm, and the development of drugs capable of alleviating cytokine storm is not easy. The virus-induced cytokine storm model is the most direct and effective choice for studying SARS-CoV-2. However, the virus research has a certain risk, especially the SARS-CoV-2 virus has strong infectivity, and has extremely high requirements on laboratory grade and personnel qualification, so the selection of the model is limited to a certain extent. There are few reports of in vivo studies in mice currently using the cytokine storm caused by SARS-CoV-2 infection directly. The non-clinical pharmacodynamics experimental method of the cytokine storm therapeutic drug comprises the steps of establishing a cytokine storm model of virus induction, LPS induction, TLR agonist induction and antigen induction on mice, and selecting primates such as rhesus monkeys for experiment more closely to the animal model of human. In vitro models, RAW264.7 cell line or primary macrophages extracted from mice, peripheral blood mononuclear cells, etc. can be selected for testing, and the induction method is also generally to stimulate monocytes or macrophages by using LPS or a TLR agonist. Typical immune cells include macrophages, dendritic cells, lymphocytes, peripheral blood mononuclear cells, and the like.

In addition, the research of SARS-CoV-2 requires a P3 laboratory, but the institutions with the P3 laboratory in the universities and enterprises of China are not more, which results in difficult direct research of SARS-CoV-2 and difficult establishment of a cytokine release syndrome model caused by SARS-CoV-2. Even if a corresponding model is established, the method is difficult to widely popularize and use due to the lack of a P3 laboratory or high cost. Due to the lack of a simple, efficient, low cost model for the massive release of SARS-CoV-2-induced cytokines, the development of drugs against SARS-CoV-2-induced cytokine release has been slow. In addition, researchers have constructed an inflammation model using alternative viruses or LPS and the like, but a model induced by non-SARS-CoV-2 cannot truly respond to the real situation that SARS-CoV-2 causes the body to release a large amount of cytokines.

Disclosure of Invention

In order to solve the above-mentioned shortcomings, the present invention aims to establish an in vitro model for large scale release of SARS-CoV-2 antigen induced cytokine without P3 laboratory, and in order to achieve the purpose, the present invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for constructing a model for inducing a cytokine storm by SARS-CoV-2 antigen, said method comprising adding SARS-CoV-2 antigen and interleukin into a culture system of immune cells.

It should be noted that the terms "comprising" and/or "including" are used to indicate that various aspects and embodiments of the disclosure "comprise" a particular feature or features. It should be understood that this/these terms may also encompass aspects and/or embodiments that "consist essentially of, or" consist of, the relevant feature or features.

As a preferred embodiment, the cell culture medium used in the immune cell culture system is a serum-free medium added to plasma.

As a preferred embodiment, the plasma is human plasma.

As a preferred embodiment, the plasma is autologous plasma, more preferably, the autologous plasma is present at a concentration of 10% in the cell culture medium.

As an alternative embodiment, the plasma may be replaced with serum, including autologous serum or fetal bovine serum.

SARS-CoV-2 is a large enveloped positive-stranded RNA virus, and the SARS-CoV-2 genome is packaged within a spiral capsid surrounded by an envelope. At least 3 structural proteins are associated with the viral envelope: spike protein (S), envelope protein (E) and membrane protein (M).

In the present invention, the SARS-CoV-2 antigen can be selected from the group consisting of: spike protein (S), envelope protein (E), membrane protein (M), nucleoprotein (N) and/or hemagglutinin esterase protein (HE).

As a preferred embodiment, the SARS-CoV-2 antigen comprises a viral antigen that is capable of acting through TLR 4.

As a preferred embodiment, the viral antigen capable of acting through TLR4 comprises spike protein.

As a preferred embodiment, the concentration of SARS-CoV-2 antigen is 0.3125, 0.625, 1.25, 2.5, 5, 10 or 20 nM, more preferably the concentration of SARS-CoV-2 antigen is 10 nM.

"Interleukin" refers to a cytokine protein produced by lymphocytes, including but not limited to cytokines commonly known as IL-2, IL-3, IL-4, IL-5, IL-10, IL-12, IL-13, IL-18, and mixtures thereof. The interleukins may be derived from natural sources or recombinantly produced.

As a preferred embodiment, the interleukin is IL-2.

As a preferred embodiment, the IL-2 concentration is 0.1-100 ng/mL, more preferably, the IL-2 concentration is 10 ng/mL.

As used herein, the term immune cell refers to any cell that plays a role in an immune response. Immune cells are derived from hematopoietic stem cells and include, but are not limited to, lymphocytes, such as T cells, NK cells, and B cells; myeloid cells such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, basophils and mast cells.

The term "lymphocyte" refers to all immature, mature, undifferentiated and differentiated lymphocyte populations, including tissue specific and specialized varieties. As non-limiting examples, it encompasses NK cells, T cells, NKT cells, and B cells.

As used herein, the term T cell includes naive T cells, CD4 ⁺ T cells, CD8 ⁺ T cells, memory T cells, activated T cells, anergic T cells, tolerating T cells, chimeric T cells, antigen-specific T cells, and regulatory T cells.

As a non-limiting example, the term "B cell (B cell or B cell)" refers to a pre-B cell, a progenitor B cell, an early pre-B cell, a late pre-B cell, a large pre-B cell, a small pre-B cell, an immature B cell, a mature B cell, a naive B cell, a plasma B cell, an activated B cell, an anergic B cell, a tolerogenic B cell, a chimeric B cell, an antigen-specific B cell, a memory B cell, a B-1 cell, a B-2 cell.

As a preferred embodiment, the immune cells are peripheral blood mononuclear cells.

As used herein, "peripheral blood mononuclear cells" refers to any cell with a single nucleus in peripheral blood, including lymphocytes and monocytes.

In a specific embodiment of the invention, the peripheral blood mononuclear cells are human peripheral blood mononuclear cells.

As a preferred embodiment, the concentration of the peripheral blood mononuclear cells is (0.5-3). Times.10 ⁶ In the specific embodiment of the invention, the concentration of the peripheral blood mononuclear cells is 1X 10 ⁶ cells/mL.

As a preferred embodiment, the method further comprises culturing the culture system with SARS-CoV-2 antigen and interleukin immune cells in a cell culture incubator.

As a preferred embodiment, the time of the culture is 4 to 24 hours, more preferably 12 to 16 hours. In a specific embodiment of the invention, the incubation time is 16 hours.

The cell culture vessel used in the culture system of the present invention is not particularly limited as long as it is capable of culturing cells, and examples thereof include culture flasks, tissue culture flasks, dishes, plates, tissue culture dishes, multi-well dishes, microplates, microwell plates, multi-well plates, culture dishes, tubes, culture bags, and roller bottles.

As a preferred embodiment, the cell culture vessel is a multi-well plate including, but not limited to, 6-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, 384-well plate. In a specific embodiment of the invention, the cell culture vessel is a 24-well plate.

In the present invention, the culture conditions can be appropriately set. For example, the culture temperature is not particularly limited, and may be about 30 to 40℃and preferably about 37 ℃. CO ₂ The concentration may be about 1 to 10%, preferably about 2 to 5%. The oxygen partial pressure may be 1 to 10%.

"cytokine", as used herein, is a widely and loosely-varying small protein (about 5-20 kDa) that is important in cell signaling. They are released by the cells and affect the behavior of other cells. Cytokines may also be involved in autocrine signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and growth factors. In specific embodiments of the invention, the cytokine comprises IL-1β, IL-6 or IL-8.

In a second aspect, the invention provides a model of SARS-CoV-2 antigen induced cytokine storm, said model being prepared by the method of the first aspect of the invention.

In a third aspect, the present invention provides the use of the model of SARS-CoV-2 antigen induced cytokine storm described in the second aspect of the present invention for screening an anti-cytokine storm drug using the model of SARS-CoV-2 antigen induced cytokine storm described in the second aspect of the present invention.

The invention has the advantages and beneficial effects that:

the invention discloses that the phenomenon that SARS-CoV-2 induces a large amount of human cytokines to be released is simulated by using SARS-CoV-2 antigen and interleukin to stimulate immune cells for the first time. The invention discloses a model of SARS-CoV-2 antigen induced cytokine storm, which has simple operation and lower requirements on laboratory level and hardware, is suitable for developing effective medicaments for resisting SARS-CoV-2 induced cytokine storm by various scientific research institutions and enterprises, is also suitable for clinically screening effective therapeutic medicaments for SARS-CoV-2 infected patients, and has good application prospect.

Drawings

FIG. 1 is a graph of experimental results of detecting the secretion of a large amount of cytokines by PBMCs stimulated by spike proteins and/or IL-2, wherein, graph A is a statistical graph of IL-1β detection results, graph B is a statistical graph of IL-6 detection results, and graph C is a statistical graph of IL-8 detection results;

FIG. 2 is a graph of experimental results for detecting cytokines in models established by different methods, wherein graph A is a statistical graph of IL-1. Beta. Detection results, graph B is a statistical graph of IL-6 detection results, and graph C is a statistical graph of IL-8 detection results;

FIG. 3 is a graph of experimental results of detecting the inhibition of cytokine storm-related inflammatory factor release by different drugs using the model of the present invention, wherein graph A is a statistical graph of IL-1β detection results, graph B is a statistical graph of IL-6 detection results, and graph C is a statistical graph of IL-8 detection results.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Experimental materials

Example construction of in vitro model of SARS-CoV-2 antigen-induced cytokine storm

Experimental method

1. Isolation of PBMCs:

1. the heparin anticoagulation tube directly collects peripheral blood (3-100 mL), extracts 0.2-1 mL blood in a biosafety cabinet for detecting mononuclear cells, T cells and NK cells in the peripheral blood by flow cytometry, distributes the rest blood into two 15 mL or 50 mL sterile centrifuge tubes on average, and centrifugates for 10 min at room temperature with 700-900 g.

2. The plasma was transferred to a new 15 mL or 50 mL sterile centrifuge tube using a disposable sterile pipette and placed in a constant temperature incubator at 56 ℃ for 30 min to inactivate complement.

3. Adding normal saline or PBS buffer solution with the same volume as the blood sampling amount into the centrifuged blood cells, uniformly mixing, slowly moving the diluted cell suspension into a 15 mL or 50 mL centrifuge tube containing lymphocyte separation liquid by adopting a disposable sterile pipette according to the proportion of 1-2:1 (volume ratio), slightly moving the centrifuge tube into the centrifuge, and centrifuging for 30 min at the room temperature at 350-450 g.

4. The centrifuge tubes were slowly removed and gently placed in a biosafety cabinet, and the middle peripheral blood mononuclear cell (Pheripheral blood mononuclear cells, PBMCs) layers were aspirated with disposable sterile pipettes and placed into new 15 mL or 50 mL sterile centrifuge tubes, each of which contained a white film layer that was not expected to exceed 10% of the tube capacity.

5. Adding PBS or physiological saline into a sterile centrifuge tube, mixing, and centrifuging at 400-500-g and room temperature for 8 min. The wash was repeated once.

2. Establishing an in vitro model

1. Modulation of PBMCs to 1X 10 ⁶ cell/mL 10% autologous plasma serum-free medium.

2. And PBMCs were seeded at 400. Mu.L/well in 24-well plates and PBS, spike protein (0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 nM, most preferably 10 nM), IL-2 (within 100 ng/mL, most preferably 10 ng/mL), IL-2 (10 ng/mL) and spike protein (10 nM) were added, respectively, at 37℃in 5% CO ₂ After incubation for 16 hours in an incubator, collecting supernatant, and temporarily storing the supernatant in a refrigerator at 4 ℃ in the detection day; can be stored for a long time and packaged, and stored in a refrigerator at-80deg.C.

3. In vitro models of SARS-CoV-2 antigen-induced cytokine storm include: (1) a PBS group; (2) spike group (10 nM); (3) IL-2 group (10 ng/mL); (4) IL-2 (10 ng/mL) in combination with spike protein (10 nM). Wherein the (1), (2) and (3) groups are used as controls, and the (4) group is an in vitro experimental model of novel coronavirus antigen induced cytokine storm.

4. ELISA or CBA detects cytokines IL-1 beta, IL-6, IL-8 and the like related to cytokine release syndrome.

3. Application (drug screening for inhibiting SARS-CoV-2 induced cytokine storm)

1. Can be used as an in vitro model for drug screening, and the experimental group comprises: (1) a PBS group; (2) spike group (10 nM); (3) IL-2 group (10 ng/mL); (4) IL-2 in combination with spike protein groups; (5) drug screening group: based on the group (4), different drugs (such as celecoxib and TLR4 inhibitor) are added, and the control effect of the different drugs on SARS-CoV-2 induced cytokine release is detected.

2. At 37 ℃,5% CO ₂ After 16 hours incubation in the incubator, the supernatant was collected. The detection on the same day can be stored in a refrigerator at 4 ℃; can be stored for a long time and packaged, and stored in a refrigerator at-80deg.C.

3. ELISA or CBA detects cytokines IL-1 beta, IL-6, IL-8 and the like related to cytokine release syndrome.

4. Based on the expression of cytokines, the anti-inflammatory effects of different drugs and the anti-inflammatory effects of different doses of drugs were analyzed.

Experimental results

1. Isolation of donor Peripheral Blood Mononuclear Cells (PBMCs), and adjustment of PBMCs to a density of 1X 10 with serum-free Medium containing 10% autologous plasma ⁶ cells/mL, 400. Mu.L/well were inoculated into 24-well plates, and PBS, spike protein (10 nM), IL-2 (10 ng/mL), IL-2-associated spike protein were then added, respectively, at 37℃with 5% CO ₂ After 16 hours incubation in the incubator, the supernatants were collected and CBA assayed for the concentration of various cytokines.

As shown in FIG. 1, the use of both spike protein and IL-2 alone stimulated Peripheral Blood Mononuclear Cells (PBMCs) to secrete IL-1 beta, IL-6 and IL-8; however, the spike protein in combination with IL-2 (10 ng/mL) was able to stimulate the secretion of more IL-1β, IL-6 and IL-8 by PBMCs. IL-1 beta, IL-6 and IL-8 are key cytokines in SARS-CoV-2 induced cytokine storm. The results in fig. 1 show that: SARS-CoV-2 spike protein, combined with lower concentrations of IL-2 to stimulate PBMCs, is able to mimic SARS-CoV-2-induced cytokine storm in vitro.

2. Isolation of donor Peripheral Blood Mononuclear Cells (PBMCs), and adjustment of PBMCs to a density of 1X 10 with serum-free Medium containing 10% autologous plasma ⁶ cells/mL, 400. Mu.L/well were inoculated into 24-well plates, and PBS, spike protein (10 nM), IL-2 (10 ng/mL), IL-2-associated spike protein, IL-15 (10 ng/mL), IL-15-associated spike protein were then added, respectively, at 37℃with 5% CO ₂ After 16 hours incubation in the incubator, the supernatants were collected and CBA detected the concentrations of IL-1. Beta., IL-6 and IL-8.

As shown in FIG. 2, IL-2 is capable of stimulating PBMCs to secrete large amounts of IL-1β, IL-6 and IL-8 in concert with spike proteins; IL-15 in combination with the spike protein stimulated PBMCs failed to produce significant amounts of IL-1β, IL-6 and IL-8 compared to the spike protein alone stimulated group.

IL-15 was selected in this study to create an in vitro model for SARS-CoV-2 drug screening because previous studies reported that IL-15, like IL-2, was able to function via IL-2 receptors β and γ chain, and that IL-15 was also significantly upregulated in SARS-CoV-2-induced cytokine storm, but this study found that IL-15 in combination with spike protein did not have significant effect in stimulating peripheral blood mononuclear cells to secrete IL-1 β, IL-6 and IL-8. The results show that the invention has good technical effect by selecting IL-2.

3. Isolation of donor Peripheral Blood Mononuclear Cells (PBMCs), and adjustment of PBMCs to a density of 1X 10 with serum-free Medium containing 10% autologous plasma ⁶ cells/mL, 400. Mu.L/well were seeded in 24 well plates, and PBS, spike protein (10 nM), IL-2 (10 ng/mL), IL-2 associated spike protein, IL-2 associated spike protein+TLR 4 inhibitor (1. Mu.M TAK-242), IL-2 associated spike protein+celecoxib (100. Mu.M), were then added, respectively, at 37℃with 5% CO ₂ After 16 hours incubation in the incubator, the supernatants were collected and CBA detected the concentrations of IL-1. Beta., IL-6 and IL-8.

As shown in fig. 3, the invention model is used for drug screening, and the inhibition effect of the TLR4 inhibitor and celecoxib on the release of inflammatory factors related to cytokine storm is detected, so that the TLR4 inhibitor and celecoxib can effectively inhibit the synergistic spike protein of IL-2 to stimulate the PBMCs to release a large amount of IL-1 beta, IL-6 and IL-8. The research results are consistent with the reported research results that the TLR4 inhibitor and celecoxib inhibitor can reduce the cytokine release caused by SARS-CoV-2, which shows that the model of the invention can be used as a drug screening for clinical treatment of novel coronary patients.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of the details are possible in light of the above teachings, and such variations are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for constructing a model of SARS-CoV-2 antigen induced cytokine storm is characterized in that the method comprises adding 10nM SARS-CoV-2 spike protein and 10ng/mL IL-2 into a culture system of human peripheral blood mononuclear cells.

2. The method of claim 1, wherein the cell culture medium used in the culture system is a serum-free medium comprising human plasma.