Method for regulating and controlling proliferation and movement of human peripheral blood T cells by Amuc1100 protein

Abstract

The invention provides a method for regulating and controlling proliferation and movement of human peripheral blood T cells by Amuc1100 protein. The method relates to the technologies of separation, activation and culture of human peripheral blood T cells, CCK-8 cell proliferation detection, Transwell cell chemotactic detection, flow cytometry receptor expression body detection and the like. The application of the Amuc1100 protein to treat T cells proves that the protein can significantly down-regulate the expression of CD25 and CXCR4 of the T cells and inhibit T cell proliferation and CXCL12 induced T cell chemotactic movement respectively. The method and research means for in-vitro regulation of T cell proliferation and chemotactic movement can be applied to targeted regulation of T cells and directional migration of intestinal tracts of the T cells, and are used for maintaining the steady state of the intestinal tracts, relieving intestinal inflammations and screening drugs to treat diseases related to the intestinal inflammations.

Description

Technical Field

[0001] The present invention relates to a method for regulating the proliferation and chemotaxis of human peripheral blood T cells in vitro by Amuc_1100 protein. Background Art

[0002] T cells are a key cell in the adaptive immune system, and their immune function and migration are crucial to intestinal homeostasis. When inflammation occurs in the body, T cells are rapidly activated and induced by chemokines released by the lesions to leave the lymph nodes and enter the blood circulation. They reach the lesions by extravasating through the blood vessel walls and exert an immune response. However, dysregulated T cell migration often leads to excessive local cell accumulation, which in turn triggers unnecessary immune responses (such as cell lytic activity and the release of proinflammatory factors), causing damage to surrounding tissues. Studies have shown that the infiltration of T cells into the intestine and their local excessive accumulation often lead to an imbalance in intestinal homeostasis, triggering chronic intestinal inflammation and even inducing inflammatory bowel disease. Therefore, rationally regulating the directional movement of T cells to the intestine and the number of T cells in the intestinal microenvironment is of great significance for maintaining intestinal homeostasis.

[0003] Amuc_1100 is an outer membrane protein of Akkermansia muciniphila (Akkermansia muciniphila). Studies in humans and mice have shown that the abundance of Amuc_1100 decreases significantly with the progression of intestinal inflammation and related diseases, such as inflammatory bowel disease. Ingestion of Amuc_1100 can alleviate intestinal inflammation and improve disease symptoms, suggesting that Amuc_1100 has a potential role in maintaining intestinal homeostasis. However, it is currently unclear whether this effect is related to Amuc_1100's direct regulation of T cells in the intestinal microenvironment. Direct studies of the effects of Amuc_1100 on T cell function, such as proliferation and chemotaxis, and the development of new experimental methods to elucidate the potential connection between the two are urgently needed. These findings have important implications for regulating intestinal homeostasis, alleviating intestinal inflammation, and treating related diseases.

[0004] CD3 is expressed on the surface of T cells and forms a protein complex with the T cell antigen receptor (T cell receptor, TCR), participating in antigen recognition and T cell activation.

[0005] CD28 is expressed on the surface of T cells and binds to the B7 molecule expressed by antigen-presenting cells, mediating T cell co-stimulatory signals and promoting T activation, proliferation and differentiation.

[0006] CD25 is the α chain of interleukin-2 receptor (IL-2R), which participates in the activation and proliferation of T cells and is often used to reflect the proliferation activity of T cells.

[0007] CXCL12, also known as SDF-1α, can induce directional T cell migration through the CXCR4 / CXCL12 chemotactic signaling pathway. CXCL12 is often used as a positive control in T cell chemotaxis assays to induce massive T cell migration. Furthermore, CXCL12 has been shown to be closely associated with the progression of intestinal inflammation, primarily through its involvement in inflammatory angiogenesis.

[0008] Peripheral blood mononuclear cells (PBMCs) are cells with a single nucleus found in peripheral blood. They primarily include lymphocytes (such as T cells, B cells, and natural killer (NK) cells) and monocytes. PBMCs develop from hematopoietic stem cells (HSCs) in the bone marrow and are a crucial component of the immune system, participating in the body's immune response and immune regulation. Summary of the Invention

[0009] The present invention directly proves through experiments that the Amuc_1100 protein can regulate the proliferation and chemotaxis of human peripheral blood T cells cultured in vitro, and thereby provides a new method for regulating the proliferation and chemotaxis of human peripheral blood T cells, providing new ideas for maintaining intestinal homeostasis, alleviating intestinal inflammation and screening drugs to treat intestinal inflammation-related diseases in the future.

[0010] By combining the application of CCK-8 cell viability assays, transwell chambers, and flow cytometry, an appropriate concentration of Amuc_1100 protein was selected to treat human peripheral blood T cells, demonstrating that Amuc_1100 protein can significantly downregulate CD25 expression in T cells and inhibit T cell proliferation; Amuc_1100 protein can significantly downregulate CXCR4 expression in T cells and inhibit CXCL12-induced T cell chemotaxis. Based on the above results, a method for regulating the proliferation and chemotaxis of human peripheral blood T cells in vitro using Amuc_1100 protein can be established. The specific operating methods of the invention are as follows:

[0011] Culture of human peripheral blood T cells (1) Isolation and activation of human peripheral blood T cells: PBMCs were isolated from human peripheral blood using density gradient centrifugation. T cells in PBMCs were selectively activated using RPMI-1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin) containing CD3 and CD28 antibodies (both 1 μg / mL) and cultured at 37°C, 5% CO2 for 48 h; (2) Cell subculture: T cell suspension was collected, centrifuged at 1000 rpm for 5 min, and then RPMI-1640 complete medium containing 12.5 ng / mL IL-2 was added to continue culturing T cells to the third generation for subsequent experiments; (3) Cell counting: After three generations of culture, the cell density was determined using a cell counting plate.

[0012] Cell proliferation detection (1) CCK-8 assay to detect T cell proliferation. The specific procedure is as follows: a. Cell inoculation and treatment: T cells were plated at 1×10 4 cells / well were seeded into 96-well plates (100 μL / well), and Amuc_1100 (5 μg / ml) was added to treat T cells, and incubated at 37°C and 5% CO2 for 36 h; b. CCK-8 assay: Refer to the instructions and add 10 μL of CCK-8 solution to each well. Incubate for 1.5 h. Measure the OD value at a wavelength of 450 nm with a microplate reader and calculate the cell proliferation rate based on the OD value. (2) Detect the expression of CD25 on the surface of T cells by flow cytometry. The specific operation method is as follows: a. Sample preparation: Take 1×10 6 The cells were centrifuged (1000 rpm, 5 min), the supernatant was discarded, and the T cells were resuspended in 100 μL PBS; b. Antibody staining: Refer to the instructions and add 5 μL of CD25-PE antibody to 100 μL of T cell suspension. Incubate at room temperature in the dark for 20 minutes. c. Washing and detection: Rinse with PBS (1 mL / time) twice and centrifuge (1000 rpm, 5 min) and resuspend in 1 mL PBS solution. CD25 expression level was detected using flow cytometry.

[0013] Cell chemotaxis assay (1) Transwell chamber was used to detect T cell chemotaxis. The specific operation method is as follows: a. Preparation of Transwell chamber: Add 100 μL of T cell solution (i.e. 2×10 5 The cells were evenly mixed in 100 μL of RPMI 1640 medium containing 1% FBS and 1% penicillin-streptomycin), and 600 μL of CXCL12 solution (i.e., 100 ng / ml CXCL12 was evenly mixed in RPMI 1640 medium containing 1% FBS and 1% penicillin-streptomycin) was added to the lower chamber; b. Incubation and detection: Incubate at 37°C and 5% CO2 for 24 hours, collect the cell suspension in the lower chamber, and count the migrated cells using a cell counter. (2) Flow cytometry was used to detect the expression of chemotactic receptor CXCR4 on the surface of T cells. The specific operation method is as follows: a. Sample preparation: Take 1×10 6 The cells were centrifuged (1000 rpm, 5 min), the supernatant was discarded, and the T cells were resuspended in 100 μL PBS; b. Antibody staining: Refer to the instructions and add 5 μL of CXCR4-PE antibody to 100 μL of T cell suspension. Incubate at room temperature in the dark for 20 minutes. c. Washing and detection: Rinse with PBS (1 mL / time) twice and centrifuge (1000 rpm, 5 min) and resuspend in 1 mL PBS solution. CXCR4 expression level was detected using flow cytometry.

[0014] Advantages and innovations of the present invention (1) For the first time, they demonstrated that Amuc_1100 protein inhibits the proliferation of human peripheral blood T cells by downregulating the expression of the T cell surface receptor CD25. This inhibitory effect on T cell proliferation can be used to alleviate the chronic intestinal inflammatory process caused by the local accumulation of unnecessary T cells. (2) For the first time, it was demonstrated that Amuc_1100 protein inhibits CXCL12-induced chemotactic movement of human peripheral blood T cells by downregulating the expression of the T cell surface chemotactic receptor CXCR4. This inhibition of CXCL12-induced T cell chemotactic movement provides new ideas for future targeted regulation of T cell intestinal migration to maintain intestinal homeostasis, alleviate intestinal inflammation, and treat intestinal inflammation-related diseases. (3) The present invention proposes and demonstrates a method for regulating the proliferation and chemotaxis of human peripheral blood T cells using Amuc_1100 protein, which provides the necessary theoretical basis for exploring more immune regulatory effects of Amuc_1100 in the future. (4) This method is based on T cell proliferation and chemotaxis, and preliminarily proves the potential regulatory effect of Amuc_1100 on them. This method provides new ideas and directions for the future screening of other intestinal probiotic-derived proteins that have the function of regulating the immune microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A: Effects of 36-h treatment of T cells with different concentrations of Amuc_1100 protein (control = 0, 0.1, 0.2, 0.5, 1, 2, 5 μg / mL) on T cell proliferation. Amuc_1100 protein at concentrations of 2 and 5 μg / mL significantly (P < 0.05) and extremely significantly (P < 0.01) inhibited T cell proliferation compared to the control group, respectively. Figure 1B: Flow cytometry analysis showed that the Amuc_1100 protein treatment group (5 μg / mL, 36 h) could significantly downregulate the expression level of CD25 on T cells compared with the control group. Figure 1 The results showed that Amuc_1100 protein could significantly inhibit the proliferation of T cells by downregulating the expression of CD25 on T cells.

[0016] Figure 2 A: Transwell chamber experiments showed that the Amuc_1100 protein treatment group (5 μg / mL, 36 h) could significantly inhibit CXCL12-induced T cell chemotaxis compared with the blank control group. Figure 2 B: Flow cytometry analysis showed that the Amuc_1100 protein treatment group (5 μg / mL, 36 h) could significantly downregulate the expression of T cell surface chemotactic receptor CXCR4 compared with the control group. Figure 2 The results showed that Amuc_1100 protein could significantly inhibit CXCL12-induced T cell chemotaxis by downregulating the expression of T cell CXCR4. DETAILED DESCRIPTION

[0017] Example The primary PBMCs used in the experiments were obtained from the Anhui Institute of Optics and Precision Mechanics, Hefei Institutes of Physical Science, Chinese Academy of Sciences. Subsequent human peripheral blood T cell culture, cell proliferation assays, cell chemotaxis assays, and flow cytometry were performed at Anhui University.

[0018] The present invention combines the use of CCK-8 cell viability assays, transwell chambers, and flow cytometry to select an appropriate concentration of Amuc_1100 protein to treat human peripheral blood T cells, demonstrating that Amuc_1100 protein can significantly downregulate CD25 expression on T cells and inhibit T cell proliferation; Amuc_1100 protein can significantly downregulate CXCR4 expression on T cells and inhibit CXCL12-induced T cell chemotaxis. Based on the above results, a method for regulating the proliferation and chemotaxis of human peripheral blood T cells in vitro using Amuc_1100 protein can be established. The detailed operating steps of the invention are as follows: (1) Draw 2-4 mL of blood using an anticoagulant blood collection tube; (2) Dilute and mix according to the ratio of 2 mL PBS to 2 mL peripheral blood (blood and PBS 1:1); (3) First, add 4 mL of lymphocyte separation solution to a 15 mL centrifuge tube, and then slowly add the diluted blood sample along the tube wall to the upper layer of the separation solution; (4) Centrifugation: 500g, 9°C (up) 1°C (down) for 20 min. After centrifugation, carefully remove the centrifuge tube. The layers are clearly separated: the upper layer is pale yellow plasma, the middle layer is milky white containing PBMCs, the lower and middle layers are transparent separation fluid, and the bottom layer is a dark red precipitate containing red blood cells. (5) Discard the upper plasma solution, slowly aspirate the PBMC from the milky white layer and transfer it to a new 15 mL centrifuge tube. Add PBS to 6 mL, centrifuge at 250 g for 10 min, discard the supernatant, and repeat the washing twice to obtain PBMC. (6) T cells in PBMCs were selectively activated using RPMI-1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin) containing CD3 and CD28 antibodies (both at 1 μg / mL) and cultured at 37°C and 5% CO2 for 48 h; (7) After 48 h, the T cell suspension was collected and centrifuged at 1000 rpm for 5 min. RPMI-1640 complete medium containing 12.5 ng / mL IL-2 was added and the T cells were cultured to the third generation for subsequent experiments.

[0019] Detection of cell proliferation and related receptor expression (1) CCK-8 cell viability assay for T cell proliferation. The specific procedure is as follows: a. T cells were cultured at 1×10 4 Cells / well were seeded into 96-well plates (100 μL / well), and blank group (i.e., no T cells, only RPMI-1640 complete medium containing IL-2), control group (T cells in RPMI-1640 complete medium containing IL-2), and Amuc_1100 concentration gradient group (i.e., T cells in RPMI1640 complete medium containing 0.1, 0.2, 0.5, 1, 2, 5 μg / mL Amuc_1100 and IL-2) were set up, and each group was repeated 3 times; b. Place the 96-well plate in an incubator at 37°C, 5% CO2 for 36 hours. Then, add 10 μL of CCK-8 solution to each well, mix thoroughly, and continue incubation for 1.5 hours. Measure the OD value at 450 nm using a microplate reader. c. Calculation of cell proliferation rate: cell proliferation rate = (OD value of experimental well - OD value of blank well) / (OD value of control well - OD value of blank well) * 100%; d. Figure 1As shown in Figure A, the effects of treating T cells with different concentrations of Amuc_1100 protein (control = 0, 0.1, 0.2, 0.5, 1, 2, 5 μg / mL) for 36 hours on T cell proliferation. Amuc_1100 protein at concentrations of 2 and 5 μg / mL had significant (P < 0.05) and extremely significant (P < 0.01) inhibitory effects on T cell proliferation, respectively, compared to the control group. (2) Flow cytometry detection of CD25 expression on T cell surface: a. Collect the T cell suspension in the T25 culture flask, count and centrifuge 1×106 cells (1000 rpm, 5 min), discard the supernatant and resuspend the T cells in 100 μL PBS; b. Refer to the instructions and add 5 μL of CD25-PE antibody to 100 μL of T cell suspension. Incubate at room temperature in the dark for 20 minutes. c. Rinse twice with PBS (1 mL / time) and centrifuge (1000 rpm, 5 min). Resuspend T cells in 1 mL of PBS solution and measure CD25 expression levels on T cells using flow cytometry. d Figure 1 As shown in B, the Amuc_1100 protein treatment group (5 μg / mL, 36 h) can significantly downregulate the expression level of T cell CD25 compared with the control group. (3) Conclusions of cell proliferation and related receptor expression detection: a. Combine Figure 1 A and Figure 1 Results B showed that Amuc_1100 protein could significantly inhibit the proliferation of T cells by downregulating the expression of CD25 on T cells.

[0020] Detection of cell chemotaxis and related receptor expression (1) Transwell chamber detection of T cell chemotaxis: a. T cells were cultured in 7 ml of medium, 5×10 5 T cells were placed at a density of 50 cells / mL in a T25 culture flask and cultured in RPMI-1640 complete medium containing 12.5 ng / mL IL-2 at 37°C and 5% CO2. A control group and an Amuc_1100-treated group were set up. The control group T cells were not treated with Amuc_1100 and cultured normally for 36 hours, while the Amuc_1100-treated group was treated with 5 μg / ml and cultured for 36 hours. b. Collect the treated T cells and add 100 μL of T cell solution (i.e. 2×10 5The cells were evenly mixed in 100 μL of RPMI 1640 medium containing 1% FBS and 1% penicillin-streptomycin), and 600 μL of CXCL12 solution (i.e., 100 ng / ml CXCL12 was evenly mixed in RPMI 1640 medium containing 1% FBS and 1% penicillin-streptomycin) was added to the lower chamber; c. Incubate at 37°C, 5% CO2 for 24 h, collect the cell suspension from the lower chamber, and count the migrated cells using a cell counter; d. Figure 2 As shown in A, the Amuc_1100-treated group could significantly inhibit the CXCL12-induced T cell chemotaxis compared with the control group. (2) Flow cytometry detection of the expression of chemotactic receptor CXCR4 on the surface of T cells: a. Collect T cell suspension in T25 culture flask, count and take 1×10 6 The cells were centrifuged (1000 rpm, 5 min), the supernatant was discarded, and the T cells were resuspended in 100 μL PBS; b. Refer to the instructions and add 5 μL of CXCR4-PE antibody to 100 μL of T cell suspension. Incubate at room temperature in the dark for 20 minutes. c. Rinse twice with PBS (1 mL / time) and centrifuge (1000 rpm, 5 min). Resuspend T cells in 1 mL of PBS solution and measure CXCR4 expression levels on T cells using flow cytometry. d. Figure 2 As shown in B, the Amuc_1100 protein treatment group (5 μg / mL, 36 h) can significantly downregulate the expression of T cell surface chemotactic receptor CXCR4 compared with the control group. (3) Conclusions of cell chemotaxis and related receptor expression: a. Combine Figure 2 A and Figure 2 Results B showed that Amuc_1100 protein could inhibit CXCL12-induced T cell chemotaxis by downregulating the expression of T cell CXCR4.

Claims

1. A method for regulating T cell proliferation and chemotaxis in vitro, characterized in that Treatment of T cells with Amuc_1100, the outer membrane protein of the probiotic Akkermansia muciniphila, can significantly inhibit T cell proliferation by downregulating the expression of the T cell surface receptor CD25; and significantly inhibit CXCL12-induced T cell chemotactic movement by downregulating the expression of the T cell surface chemotactic receptor CXCR4.

2. The method according to claim 1, characterized in that After treating peripheral blood T cells cultured in vitro with 5 μg / mL of Amuc_1100 for 36 hours, the proliferation and chemotaxis of T cells were detected. The specific procedures are as follows: (1) PBMCs were isolated from human peripheral blood using a lymphocyte separation medium and density gradient centrifugation. T cells in PBMCs were selectively activated using RPMI-1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin) containing CD3 and CD28 antibodies (both at 1 μg / mL) and cultured at 37°C and 5% CO2 for 48 h. (2) The T cell suspension was collected and centrifuged at 1000 rpm for 5 min. RPMI-1640 complete medium containing 12.5 ng / mL IL-2 was added and the T cells were cultured to the third generation for subsequent experiments. (3) After three generations of culture, the cell density was determined using a cell counting plate; (4) T cells (cell density 5×10 5 cells / mL) for 36 h, and then CCK-8 cell viability assay, Transwell chamber and flow cytometry were used to detect cell proliferation and chemotaxis.

3. The method according to claim 2, characterized in that After treating T cells with Amuc_1100 (5 μg / mL) for 36 hours, the proliferation of T cells and the expression of T cell surface receptor CD25 were detected by CCK-8 and flow cytometry, respectively, which proved that the inhibitory effect of Amuc_1100 on T cell proliferation was achieved by downregulating the expression of T cell surface receptor CD25. The chemotactic movement of T cells induced by CXCL12 and the expression of T cell surface chemotactic receptor CXCR4 were detected by Transwell chamber experiment and flow cytometry, respectively, which proved that the inhibitory effect of Amuc_1100 on CXCL12-induced T cell chemotactic movement was achieved by downregulating the expression of T cell surface chemotactic receptor CXCR4.

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