Use of lysophosphatidylcholine in promoting b-cell proliferation

Abstract

The application discloses application of lysophosphatidylcholine in promoting B cell proliferation, wherein the lysophosphatidylcholine is LysoPC 18:0, the chemical formula is C 26 H 54 NO7P, and the structural formula is The application finds through animal experiments that LysoPC 18:0 has a significant effect of promoting B cell proliferation, can be used for preparing a reagent for promoting B cell proliferation in vitro or in vivo, provides a basis for diagnosing autoimmune diseases such as lupus erythematosus, scleroderma, Sjogren's syndrome and vasculitis, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to the application of lysophosphatidylcholine in promoting B cell proliferation, particularly the application of lysophosphatidylcholine 18:0 (LysoPC 18:0) in promoting B cell proliferation. It should be noted that this invention is a divisional application based on patent application number 202210671127.0. Background Technology

[0002] Follicular helper T (Tfh) cells are a subset of CD4 T cells mainly found in germinal centers. They are the main CD4 T cells that maintain and promote the proliferation and differentiation of germinal center B cells into plasma cells and memory B cells. A significant characteristic of Tfh cells is the expression of molecules such as CD4, CXCR5, PD-1, and IL-21. Among these, IL-21 is the most important cytokine for Tfh cell function, although Tfh cells do not always secrete IL-21. IL-21 is the most potent cytokine promoting plasma cell differentiation and antibody affinity maturation, and it is also an essential cytokine for germinal center B cell differentiation. Studies have shown that the number and percentage of Tfh cells are increased in patients with systemic lupus erythematosus (SLE), and this percentage is positively correlated with antibody titers, lupus disease activity, and the number of circulating plasmablasts. Research has also reported that Tfh cells are involved in the pathogenesis of other autoimmune diseases such as rheumatoid arthritis, scleroderma, Sjögren's syndrome, and vasculitis. However, Tfh cells are extremely rare in the human body. Detecting the IL-21 secretion function of Tfh cells in humans requires a large amount of peripheral blood, making it even more difficult to isolate enough Tfh cells for in vitro studies. Therefore, it is particularly important to find new indicators reflecting the functional Tfh cells and to establish in vitro Tfh cell expansion systems.

[0003] Lysophosphatidylcholine (LysoPC) is an immunologically active inflammatory lipid molecule and a major component of oxidized low-density lipoprotein. It is primarily produced by the hydrolysis of phosphatidylcholine by phospholipase A2. LysoPC can induce the migration of lymphocytes and macrophages and increase the secretion of inflammatory cytokines. Studies have shown that LysoPC can promote the secretion of IL-1β, IL-6, and TNF-α by adipocytes and the secretion of IFN-γ and TNF-α by lymphocytes. Currently, there are no reports of LysoPC 18:0 promoting the secretion of IL-21 by Tfh cells or promoting B cell proliferation. Summary of the Invention

[0004] The first objective of this invention is to provide the use of LysoPC 18:0 in the preparation of a secretion promoter for IL-21 secretion by Tfh cells in vitro or in vivo.

[0005] Preferably, the in vitro cell intervention concentration of LysoPC 18:0 is 5-30 μM, and the in vivo cell intervention concentration is 5-50 mg / Kg.

[0006] A second object of the present invention is to provide the use of LysoPC 18:0 in the preparation of reagents that promote B cell proliferation in vitro or in vivo.

[0007] Preferably, the in vitro cell intervention concentration of LysoPC 18:0 is 5-30 μM, and the in vivo cell intervention concentration is 5-50 mg / Kg.

[0008] A third objective of this invention is to provide the application of LysoPC 18:0 in the preparation of reagents for diagnosing autoimmune diseases associated with Tfh cells.

[0009] Preferably, the Tfh cell-related autoimmune diseases include lupus, scleroderma, Sjögren's syndrome, and vasculitis.

[0010] Preferably, the reagent is used to detect the level of LysoPC 18:0 in vitro as an indicator to assess the disease activity of the Tfh cell-related autoimmune disease.

[0011] This invention is the first to discover that LysoPC 18:0 can significantly promote IL-21 secretion by Tfh cells and also promote B cell proliferation, both in vivo and in vitro. Therefore, it can be used to prepare secretion promoters of IL-21 from Tfh cells in vitro or in vivo, or reagents to promote B cell proliferation in vitro or in vivo. Furthermore, LysoPC 18:0 levels are higher than IL-21 levels, and it is more sensitive and easily measurable than Tfh cells. High levels of LysoPC 18:0 can reflect the presence of Tfh cells with high IL-21 secretion. Detecting LysoPC 18:0 levels can be used as a substitute for reflecting Tfh cells' IL-21 secretion function, and can be used to diagnose autoimmune diseases related to Tfh cells, such as lupus erythematosus, scleroderma, Sjögren's syndrome, and vasculitis. It also provides a foundation for subsequent research on the mechanism of action of IL-21-secreting Tfh cells and the establishment of animal models of Tfh cells with high IL-21 secretion. Attached Figure Description

[0012] Figure 1 In this example, flow cytometry was used to detect the effect of LysoPC 18:0 on the IL-21 secretion function of Tfh cells induced to differentiate in vitro. A: Flow cytometry results showed that LysoPC 18:0 promoted the secretion of IL-21 by Tfh cells; B: Statistical graph of results A. The comparison between the means of two independent samples was performed using a t-test, and ** indicates P < 0.01.

[0013] Figure 2To illustrate the effect of LysoPC 18:0 on the IL-21 secretion function of Tfh cells in vivo using flow cytometry, A: Flow cytometry results showed that LysoPC 18:0 promoted the secretion of IL-21 by CD4 T cells in vivo; B: Statistical graph of results A. The comparison between the means of two independent samples was performed using a t-test, where * indicates P < 0.05 and ** indicates P < 0.01.

[0014] Figure 3 To investigate the effect of LysoPC 18:0 on B cell proliferation in vivo using flow cytometry in this embodiment, A: Flow cytometry results showed that LysoPC 18:0 promoted the proliferation of CD19+ B cells in vivo. + Proliferation; B: Statistical graph of results A. The comparison between the means of two independent samples was performed using a t-test, and ** indicates P < 0.01. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] The LysoPC 18:0,1-octadecyl-sn-glycerol-3-phosphorylcholine used in the following examples has the chemical formula: C 26 H 54 NO7P, the structural formula is as follows:

[0017] Purchase purified LysoPC 18:0 from Sigma. Commercial LysoPC 18:0 is dissolved in 1% ethanol-distilled water to prepare a 100 mM stock solution.

[0018] Example 1

[0019] This embodiment verifies the effect of LysoPC 18:0 on the IL-21 secretion function of in vitro induced Tfh cells through human peripheral blood cell experiments. The procedure is as follows: Human peripheral blood was collected, and peripheral mononuclear cells (PBMCs) were separated using gradient density centrifugation. CD4 T cells were then enriched using Stemcell's EasySep Human CD4 T Cell Iso Kit. LysoPC 18:0 stock solution was added during the in vitro Tfh differentiation process. The IL-21 secretion function of Tfh cells was then measured by flow cytometry. The specific steps are as follows:

[0020] (1) Gradient density centrifugation method for separating peripheral mononuclear cells

[0021] ① Preparation of stock solution for commercial LysoPC 18:0: Dissolve in 1% ethanol-distilled water to prepare a 100mM stock solution.

[0022] ② Gradient density centrifugation for peripheral blood PBMC separation: Add 3-4 mL of lymphocyte separation medium to a new empty 15 mL centrifuge tube. Add 3 times the volume of physiological saline to 6 mL of anticoagulated peripheral blood and mix well. Then gently add the peripheral blood to the centrifuge tube containing the lymphocyte separation medium, being careful not to disturb the lymphocyte separation medium layer. Set the centrifuge to slow rise and slow fall, and centrifuge at 3500 rpm for 25 minutes.

[0023] ③ After centrifugation, the following layers can be observed: the lower layer consists of red blood cells and granulocytes, the middle layer consists of lymphocyte separation fluid and mononuclear cells, and the upper layer consists of physiological saline. Carefully aspirate the middle mononuclear cell layer, add it to a new centrifuge tube, add 10 mL of physiological saline, and mix well.

[0024] ④ Centrifugation: Centrifuge at 500g for 5 minutes, then discard the supernatant.

[0025] ⑤ Wash twice: Add 10mL of physiological saline, mix well by pipetting, centrifuge at 500g for 5 minutes and discard the supernatant. Repeat once. The remaining cell clusters are PBMCs, which can be used for subsequent experiments.

[0026] (2) Isolation of human peripheral blood CD4 T cells

[0027] Human peripheral blood CD4 was isolated using Stemcell's Human CD4 T Cell Isolation Kit. + T cells, specifically as follows:

[0028] ① Resuspend cells: Add EasySep Buffer to the PBMC cell clusters to resuspend the cells, adjusting the cell concentration to 5 × 10⁻⁶. ^7 Cells / mL, and the cell suspension was transferred to a sterile flow cytometer.

[0029] ② Antibody incubation: Add Isolation Cocktail to the cell suspension at a ratio of 50 μL / mL, mix gently, and incubate at room temperature for 5 minutes.

[0030] ③ Add sorting magnetic beads: Vortex RapidSphere for 30 seconds, add cell suspension at a ratio of 50 μL / mL, and add EasySep Buffer to 2.5 mL. Gently pipette and mix 2-3 times, and place the flow cytometer into the sorting magnetic frame.

[0031] ④Incubation: Incubate at room temperature for 3 minutes.

[0032] ⑤ Sorting: Pick up the magnetic rack and pour the liquid from the flow cytometry tube into a new 15mL centrifuge tube. Then perform cell counting.

[0033] ⑥ Cell culture: After centrifugation at 400g for 5 minutes, discard the supernatant, add cell culture medium, and resuspend the cells at a density of 5×10⁻⁶. ^5 per mL.

[0034] (3) In vitro induction and differentiation of human Tfh cells

[0035] The cells were divided into two groups: a control group and a LysoPC 18:0 group. The LysoPC 18:0 group was treated with LysoPC 18:0 at a final concentration of 10 μM, while the control group was treated with an equal volume of 1% ethanol-distilled water. Cytokines were then added to both groups of cells to induce differentiation into Tfh cells. After 3 days of induction, the medium was changed and the cells were divided into wells at a ratio of 1:2 or 1:3. Cells were collected after 5 days for subsequent experiments, and the experiment was repeated 4 times.

[0036] The components used to induce Tfh cell differentiation are: culture medium: ImmunoCult TM -XF T Cell Expansion Medium (purchased from Stemcell, #10981), β-mercaptoethanol: 50 μM, ImmunoCult TM Human CD3 / CD28 / CD2T Cell Activator (Stemcell, #10970): 20 μL / mL, activin A: 50 ng / mL, IL-12: 5 ng / mL, TGF: 5 ng / mL, IL-23: 10 ng / mL. All cytokines were purchased from PeproTech.

[0037] (4) Flow cytometry detection of Tfh secretion of IL-21

[0038] Add cell stimulant cocktail (Invitrogen, #00-4970-03) to the culture wells and incubate for 5 hours. Collect the stimulated cells into flow cytometry tubes, wash once with 2 mL of flow cytometry staining solution, centrifuge at 400g for 5 minutes, and discard the supernatant, as follows:

[0039] ① Antibody incubation: Add 0.5 μL Fixable Viability Dye eFlour 450, 2 μL anti-human CD4-FITC, anti-human CXCR5-APC, and anti-human PD-1-PE flow cytometry antibodies to 100 μL flow cytometry staining solution to resuspend the cell pellet, and incubate at room temperature in the dark for 30 minutes.

[0040] ② Washing: Add 2 mL of flow cytometry staining solution to resuspend the cells, centrifuge at 400 g for 5 minutes, and discard the supernatant.

[0041] ③ Fixation: Add 200 μL of IC Fixation Buffer (purchased from Invitrogen, #00-8222-49) to the flow cytometer and fix at room temperature in the dark for 20 minutes.

[0042] ④ Membrane rupture: Add 3 mL of 1×Permeabilization Buffer (purchased from Invitrogen, #00-8333-56) to the flow cytometer, centrifuge at 400g for 5 minutes, discard the supernatant, and repeat the washing once.

[0043] ⑤ Antibody incubation: Add 2 μL of anti-human IL-21-percp-cy5 flow cytometry antibody to 100 μL of 1×Permeabilization Buffer to resuspend the cell pellet, and incubate at room temperature in the dark for 30 minutes. Add 3 mL of 1×Permeabilization Buffer to the flow cytometry tube, centrifuge at 400g for 5 minutes, discard the supernatant, and repeat the washing once.

[0044] ⑥ Resuspension assay: Add 500 μL of flow cytometry staining solution to resuspend the cell pellet and detect it using a BD AriaIII flow cytometer.

[0045] The results are as follows Figure 1 As shown, LysoPC 18:0 has the ability to promote Tfh cell (CD4+) cell growth in vitro. + CXCR5 + PD-1 + The role of secreting IL-21.

[0046] Example 2

[0047] This embodiment verifies the promoting function of LysoPC 18:0 on IL-21 secretion by Tfh cells and B cell proliferation in mice through intraperitoneal injection of LysoPC 18:0 or saline. The procedure is as follows: Mice were injected intraperitoneally with LysoPC 18:0 or saline. Peripheral blood was collected at 12 and 24 hours later for flow cytometry analysis to detect the function of CD4 T cells in secreting IL-21 and the percentage of B cells. The specific steps are as follows:

[0048] Ten healthy adult C57 / BL6 mice aged 8 weeks were randomly divided into two groups of 5 mice each. Each group was injected intraperitoneally with 200 μL of LysoPC 18:0 (50 mg / Kg) or 200 μL of physiological saline. The mice were euthanized at 12 hours and 24 hours. Peripheral anticoagulated blood was collected from the mice for subsequent experiments. The experiment was repeated 3 times.

[0049] Flow cytometry was used to detect the IL-21 secretion function of Tfh cells and the number of B cells in mouse peripheral blood, as follows:

[0050] ① Cracked Red: Add 5 mL of cracked red solution and incubate at room temperature for 10 mL.

[0051] ② Washing: Add 20mL of physiological saline, centrifuge at 400g for 5 minutes, and then wash twice.

[0052] ③ Stimulation: Resuspend cells in RPMI 1640 cell culture medium at 1.5 × 10⁶ cells / day. ^6 Cells / mL, cell stimulant cocktail, stimulated in a cell culture incubator for 5 hours.

[0053] ④ Flow cytometry staining: Collect cells in flow cytometry tubes, wash once with 2 mL of flow cytometry staining solution, add flow cytometry antibody, 0.5 μL Fixable Viability Dye eFlour 450, 2 μL anti-mouse CD4-FITC, anti-mouse CXCR5-APC, anti-mouse PD-1-PE, and anti-mouse CD19-percp-cy5 flow cytometry antibodies, and resuspend the cell pellet in 100 μL of flow cytometry staining solution. Incubate at room temperature in the dark for 30 minutes.

[0054] ⑤ Washing: Resuspend the cells in 2 mL of flow cytometry staining solution, centrifuge at 400 g for 5 minutes, and discard the supernatant.

[0055] ⑥ Fixation: Add 200 μL of IC Fixation Buffer to the flow cytometer and fix at room temperature in the dark for 20 minutes.

[0056] ⑦ Membrane disruption: Add 3 mL of 1×Permeabilization Buffer to the flow cytometer, centrifuge at 400 g for 5 minutes, and discard the supernatant. Repeat the washing once.

[0057] ⑧ Antibody incubation: Add 2 μL of anti-mouse IL-21-PE flow cytometry antibody to 100 μL of 1×Permeabilization Buffer to resuspend the cell pellet, and incubate at room temperature in the dark for 30 minutes.

[0058] Add 3 mL of 1×Permeabilization Buffer to a flow cytometry tube, centrifuge at 400 g for 5 minutes, and discard the supernatant. Repeat the washing once, resuspend the cell pellet in 500 μL of flow cytometry staining solution, and analyze using a BD Aria III flow cytometer.

[0059] The results are as follows Figure 2 and 3 As shown, LysoPC 18:0 promotes the secretion of IL-21 by Tfh cells in vivo. Figure 2 LysoPC 18:0 also showed that it promotes the growth of B cells (CD19) in vivo. + Proliferation ( Figure 3 ).

[0060] In summary, LysoPC 18:0, with in vitro cell intervention concentrations of 5-30 μM and in vivo intervention concentrations of 5-50 mg / Kg, significantly promotes IL-21 secretion by Tfh cells and also significantly promotes B cell proliferation, exhibiting this effect both in vivo and in vitro. Furthermore, LysoPC 18:0 levels are higher than IL-21, and it is more sensitive and easily detectable than Tfh. It can be detected using conventional methods familiar to relevant technicians, such as high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), HPLC-electrospray ionization tandem mass spectrometry (HPLC-EFFI / MS), and high-throughput EFFI / MS. Detection of LysoPC 18:0 levels can be used as an alternative indicator of Tfh IL-21 secretion function for the diagnosis of Tfh cell-related autoimmune diseases such as lupus erythematosus, scleroderma, Sjögren's syndrome, and vasculitis.

[0061] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Application of LysoPC 18:0 in the preparation of reagents for promoting B cell proliferation in vitro, wherein the chemical formula of LysoPC 18:0 is C 26 H 54 NO7P, the structural formula is: 。 2. The application according to claim 1, characterized in that, The in vitro cell intervention concentration of LysoPC 18:0 was 5-30 μM.

Images