Application of LRG1 in preparation of medicine for promoting differentiation of macrophages to M1 type and macrophage activator

Abstract

The invention relates to application of LRG1 in preparation of a medicine for promoting differentiation of macrophages to M1 type and a macrophage activator. The invention finds that the LRG1 activates the macrophages and induces the macrophages to differentiate to the pro-inflammatory M1 type, so that the LRG1 is a new inducible factor activated by the macrophages. The invention provides a new mechanism for macrophage activation, and provides a new target spot and thought for preventing and treating inflammatory reaction and inflammatory reaction related diseases and tumors.

Description

Technical Field

[0001] The present invention relates to the use of LRG1 in the preparation of a drug for promoting macrophage differentiation into M1 type and a macrophage activator. More specifically, the present invention relates to LRG1 promoting macrophage differentiation into M1 type, thereby being used as a macrophage activator for promoting inflammatory response against microorganisms or for anti-tumor. Background Art

[0002] Macrophages participate in multiple physiological processes such as maintaining homeostasis and metabolic regulation, and also participate in forming the "first line of defense" of the human body against external danger signals. Under normal physiological conditions, macrophages are "scavengers" in the body, and play an important physiological function in maintaining the homeostasis of tissues and organs by stabilizing the microenvironment of tissue cells, cleaning metabolic wastes, and removing damaged and diseased cells. Monocytes in the body's blood circulation system are macrophage precursor cells widely recognized by the academic community. Under special stimulation, they can differentiate into mature macrophages and reach different tissues and organs with the blood circulation system to function. Under pathological conditions, macrophages and other immune cells jointly build the "first line of defense" against external danger signals. On the one hand, macrophages can actively identify pathogens and phagocytose them to prevent the spread of pathogens in the body; on the other hand, macrophages are important antigen presenting cells, which directly activate the body's "cellular immune response" to promote a stronger immune response. In summary, macrophages play a vital role in both innate immunity and acquired immunity.

[0003] Macrophages are mainly divided into two categories, namely the M1 type that promotes inflammation and the M2 type that inhibits inflammation. Functionally, M1 macrophages can produce proinflammatory cytokines such as TNFα (tumor necrosis factor α), IL-1α, IL-1β, IL-6, etc., which inhibit the proliferation of surrounding cells. A large number of studies have shown that TNFα, IL-6, IL-1α and IL-1β secreted by M1 macrophages can effectively enhance the killing effect of macrophages on tumors and promote the opening of tumor cell necrosis and apoptosis pathways. M1 macrophages participate in the clearance of pathogens during infection by activating the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system and subsequently producing reactive oxygen species (ROS). As a result, M1 macrophages can promote inflammatory responses, have a strong ability to kill microorganisms and tumor cells, and play an important role in killing intracellular infected pathogens and anti-tumor immunity. In particular, M1 macrophages express high levels of histocompatibility complexes and co-stimulatory factors, such as CD80 and CD86, which can further activate the adaptive immune system to kill tumor cells. In contrast, M2 macrophages mainly secrete anti-inflammatory cytokines such as IL-10, a variety of chemokines and growth factors to inhibit inflammatory responses, and secrete cytokines that can stimulate the expression of extracellular matrix and accelerate tumor angiogenesis, leading to tumor growth and metastasis.

[0004] Therefore, polarizing macrophages into M1 macrophages that inhibit cancer development has become the key to improving immunotherapy for solid tumors.

[0005] Leucine-rich α2-glycoprotein 1 (LRG1) is a protein encoded by the gene LRG1, which is secreted by the liver and is also a highly conserved member of the leucine-rich repeat (LRR) family of proteins. It is mainly involved in cell adhesion, migration, and signal transduction, and is a newly discovered angiogenic factor in recent years. LRG1 is expressed during granulocyte differentiation. LRG1 participates in promoting angiogenesis by causing the conversion of transforming growth factor β (TGFbeta) signals in endothelial cells).

[0006] Wang et al.[1] found that compared with normal mice, LRG1 expression was significantly increased in the retinal neovascularization model. LRG1 activated the TGF-β1 signal transduction pathway by binding to its co-receptor ENG, shifting the TGF-β signal from maintaining vascular stability to a pathological angiogenesis state, activating the pro-angiogenic signal TβR-Ⅱ, and then recruiting endothelial ALK-1, initiating the phosphorylation of transcription factors Smad 1 / 5 / 8, promoting VEGF transcriptional upregulation and increased expression, and then promoting endothelial cell migration and proliferation, thereby promoting pathological angiogenesis.

[0007] However, no one has yet found that LRG1 is associated with macrophages. Summary of the invention

[0008] The inventors discovered the relationship between LRG1 and macrophages. Specifically, by stimulating mouse bone marrow-derived macrophages (BMDM) with LRG1, it was found that LRG1 can promote macrophage activation. The inventors further used magnetic bead sorting to analyze the changes in cytokines in the cell culture medium after LRG1 stimulation, and found that LRG1 can significantly promote the secretion of macrophage inflammatory factors. M1 and M2 macrophages were marked by CD80 and CD206, respectively, and flow analysis was used to find that macrophages differentiated into pro-inflammatory M1 phenotypes after LRG1 intervention. Therefore, it is concluded that LRG1 promotes the differentiation of macrophages into M1 type, thereby forming the present invention.

[0009] In one aspect, the present invention provides use of LRG1 in preparing a drug for promoting differentiation of macrophages into M1 type.

[0010] In one embodiment, the differentiation of macrophages into M1 type is manifested by one or more of the following:

[0011] (1): enhancing the secretion of one or more inflammatory factors selected from TNFα, VCAM-1, MCP-1, IL-6, IL-1α and IL-1β, in particular TNFα and / or VCAM-1;

[0012] (2): Increased expression of CD80;

[0013] (3): Enhance the adhesion of macrophages to tumor cells.

[0014] In one embodiment, the medicament is used to promote an inflammatory response against a microorganism or for anti-tumor effects, ie, for treating or preventing a bacterial or viral infection or for treating or preventing a tumor.

[0015] In one embodiment, the tumor is a solid tumor, such as breast cancer, ovarian cancer, cervical cancer, melanoma, or prostate cancer.

[0016] In one embodiment, the microorganism is a bacterium or a virus.

[0017] In another aspect, the present invention provides a macrophage activator comprising LRG1 as an active ingredient and promoting differentiation of macrophages into M1 type.

[0018] In one embodiment, the macrophage activator is used to promote an inflammatory response against a microorganism or for anti-tumor effects.

[0019] In one embodiment, the tumor is a solid tumor, such as breast cancer, ovarian cancer, cervical cancer, melanoma, or prostate cancer.

[0020] In one embodiment, the microorganism is a bacterium or a virus.

[0021] Preferably, the macrophages are derived from bone marrow. This is because the content of macrophages in the blood is very small, and most of them are derived from bone marrow. At present, bone marrow is generally recognized by the academic community as the birthplace of macrophages. Therefore, taking macrophages from bone marrow is the easiest way to obtain macrophages in large quantities in experiments. The macrophages obtained by this method can generally represent the common characteristics of macrophages and serve as a universal template for studying macrophages.

[0022] As used herein, in the present invention, prevention or treatment has a common meaning and should refer to the beneficial effects of the subject derived from treatment. In certain embodiments, at least one of the following effects is achieved by prevention or treatment, including, for example: (i) reducing or improving the severity of the disease or symptoms associated therewith; (ii) reducing the duration of symptoms associated with the disease; (iii) preventing the progression of the disease or symptoms associated therewith; (iv) the regression of the disease or symptoms associated therewith; (v) preventing the development or onset of symptoms associated with the disease; (vi) preventing the recurrence of symptoms associated with the disease; (vii) reducing the hospitalization of the subject; (viii) reducing the length of hospitalization; (ix) the survival of subjects with the disease increases; (x) reducing the number of symptoms associated with the disease; (xi) one or more preventive effects or therapeutic effects of another therapy are enhanced, improved, supplemented, complemented or increased. Administration can be by a variety of routes, including but not limited to intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and / or local delivery to the affected tissue.

[0023] The present invention found that LRG1 activates macrophages and induces macrophages to differentiate into pro-inflammatory M1 type, so LRG1 is a new inducing factor for macrophage activation. The present invention provides a new mechanism for macrophage activation, and provides new targets and ideas for the prevention and treatment of inflammatory response, inflammatory response-related diseases and tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of the in vitro induction of primary macrophage M1 / M2 differentiation experiment are shown. It is divided into four groups: blank control group, M1 macrophage induction group, M2 macrophage induction group and LRG1 stimulation group. The blank control group does not add any substance, and the latter three are stimulated with LPS+IFNγ, IL-4 and LRG1 respectively. The results show that LRG1 promotes the transformation of macrophages to M1 phenotype, among which the control group; LPS+IFNγ; IL-4; LRG1 groups respectively stimulate BMDM and change the cell morphology.

[0025] Figure 2The results show that flow cytometry was used to detect the comparison of CD80, a surface marker of M1 macrophages, in vitro induced differentiation of primary macrophages. The results showed that LRG1 promoted the transformation of macrophages to M1 phenotype. Flow cytometry was used to mark M1 surface markers with CD80, and the effect of LRG1 on macrophage phenotype was identified. After LRG1 stimulated macrophages, the macrophage phenotype was more inclined to M1.

[0026] Figure 3 The results show that flow cytometry was used to detect the comparison of CD206, a surface marker of M2 macrophages, in vitro induced differentiation of primary macrophages. The results showed that LRG1 promoted the transformation of macrophages to M1 phenotype. Flow cytometry was used to mark M2 surface markers with CD206 to identify the effect of LRG1 on macrophage phenotype. After LRG1 stimulated macrophages, the macrophage phenotype was more inclined to M1.

[0027] Figure 4 The protein concentration curves of six cellular inflammatory factors with anti-tumor properties secreted by macrophages under stimulation with different concentrations of LRG1 are shown.

[0028] Figure 5 It showed that LRG1 could enhance the adhesion ability of macrophages to tumor cells induced by oxLDL. DETAILED DESCRIPTION

[0029] Example 1 Study on the effect of LRG1 on macrophage phenotypic differentiation

[0030] Bone marrow-derived macrophage (BMDM) culture

[0031] 1) 6-12 week old C57BL / 6c mice were killed by cervical dislocation and sprayed with 70% medical alcohol for 3-5 minutes;

[0032] 2) Pour 5 ml of D-Hanks solution into a 35 mm diameter culture dish and place the dish on an ice pack to cool;

[0033] 3) Take out the sterilized mice and place them in a culture dish;

[0034] 4) Open the sterile microsurgical instrument package, take out the ophthalmic scissors and tweezers, adjust the mouse to a supine position, cut the skin along the line of the mouse's hind paw at the hip joint of the mouse's thigh, and use tweezers to peel off and remove subcutaneous fat, muscle, nerve and other tissues in sequence to fully expose the femur and tibia and fibula. Use scissors to carefully shorten the connection between nerves, muscles and bones, and separate the femur and tibia and fibula at the hip joint and ankle joint respectively. Be careful not to expose the bone marrow cavity here.

[0035] 5) Take an ice box, soak the removed femur and tibia and fibula in a 15 ml centrifuge tube containing 70% medical alcohol, and place it on the ice box.

[0036] 6) Wash the alcohol off the surface of the femur and tibia and fibula with ice PBS, insert a 10mL syringe connected to a 1mL syringe needle into the bone marrow cavity, and quickly inject the previously drawn ice PBS solution into the bone marrow cavity to flush out the bone marrow. Repeat three times to ensure that the bone marrow tissue of the mouse hind limb is fully obtained;

[0037] 7) Filter the removed bone marrow tissue with a 40 μm filter, and grind the remaining large pieces of bone marrow repeatedly to obtain sufficient bone marrow cells. Use a 10 ml pipette to repeatedly blow the PBS solution containing the bone marrow tissue to mix it evenly.

[0038] 8) Transfer the PBS liquid containing bone marrow cells to a 15 mL centrifuge tube with a pipette, centrifuge at 4°C, 900 g, for 4 min, discard the supernatant, add 6-10 mL of red blood cell lysis buffer, resuspend, let stand at room temperature for 3-5 min, and centrifuge at 4°C, 900 g, for 4 min.

[0039] 9) Take out the centrifuged cells, discard the supernatant, and resuspend and culture them in DMEM medium containing 10-20% L929 cell culture medium supernatant, 10% FBS, and 1% penicillin / streptomycin antibiotics.

[0040] LRG1 induction intervenes in macrophages and polarization induction and identification of M1 and M2 phenotypes

[0041] Stimulation induction:

[0042] The above cultured macrophages were cultured until the 6th day and then divided into plates. The cells were plated at 5*10 5 -1*10 6 The cells were seeded at a density of 100 μg / mL in a 6-well plate and incubated overnight at 37°C. LPS (1 μg / mL) + IFNγ (50 ng / mL) IL4 (10 ng / mL); LRG1 (100 ug / mL) was added to the cells to stimulate the macrophages for 24 hours, and then the cells were divided into the following groups: M0, M1 group (LPS + IFNγ), M2 (IL-4) group and LRG1 group, namely blank control group, M1 macrophage induction group, M2 macrophage induction group and LRG1 stimulation group. The cells were photographed separately, as shown in Figure 2. Figure 1 As shown, keep the cells for later use.

[0043] 2) Phenotypic identification:

[0044] The expression of macrophage markers F4 / 80, M1 macrophage marker CD80, and M2 macrophage marker CD206 was detected by flow cytometry (the specific steps are the same as in 2.2 below) to determine that mature M1 and M2 macrophages were obtained and to identify the induced phenotype of M0 macrophages by LRG1.

[0045] Flow cytometry was used to detect the expression of CD80 and CD206

[0046] 2.2 Flow cytometry detection of cell CD80 expression

[0047] 1) Take out the cells after overnight intervention from the incubator, remove the conditioned medium in the culture dish, wash with PBS, add trypsin to digest for 10 minutes, and use DMEM solution prepared with 100mL / L FBS to terminate the digestion. Use a pipette to transfer the suspended cells to a 15mL centrifuge tube and centrifuge at 500g for 4 minutes.

[0048] 2) After centrifugation, discard the supernatant of the cells, add 1 mL of MACS solution to resuspend and wash 1-2 times, centrifuge at 4°C, 500g for 4 minutes, discard the supernatant, add 100 μL FC block (CD16 / 32) to each sample, and block on ice for 5 minutes. The samples were divided into two groups: A-CD80 detection group; B-CD206 detection group

[0049] 3) Group A: Place each sample in a 1.5mL EP tube, add F4 / 80-APC and CD80-PE antibodies at a concentration of 1:400, dilute the antibodies with FC-block, set up single positive tubes, F4 / 80-APC, CD80-PE, and incubate at 4°C for 30min-1h. Group B: Place each sample in a 1.5mL EP tube, set up control tubes, single positive tubes, and test tubes: blank control tube, F4 / 80-APC, CD206-BV421, F4 / 80-APC+CD206-BV421 tube, add F4 / 80-APC antibodies at a concentration of 1:400, dilute the antibodies with FC-block, and incubate at 4°C for 30min-1h.

[0050] 4) Antibody washing: Centrifuge group A, discard the supernatant, resuspend the antibody 1-2 times in 100-200μL MACS buffer (PBS can be substituted), resuspend in 100μL MACS buffer, add DAPI (1:1000) for nucleus staining. If stored, resuspend in PFA. Centrifuge group B, discard the supernatant, resuspend the antibody 1-2 times in 100-200μL MACS buffer (PBS can be substituted), add fix / perm (invitrogen eBioscience TM foxp3 / Transcription) was fixed at room temperature for 1 hour, and then washed 1-2 times with Permeabilization Buffer. Then, the cells were stained with antibody diluted in buffer (1:400) for 30 minutes to 1 hour, centrifuged, washed 1-2 times with buffer, and resuspended in 300 μL MACS before loading.

[0051] 5) The cells were transferred to a flow cytometer and the expression of cell surface markers was detected by flow cytometry.

[0052] The experimental results are shown in Figure 1-Figure 3 middle.

[0053] Depend on Figure 1 It can be seen that LRG1 promotes the differentiation of macrophages into M1 macrophages.

[0054] Specifically, in the blank control group, the macrophages cultured in vitro were mostly plump and round in morphology, with obvious decomposition of the nucleus and cytoplasm, and the cell body was plump and spread out to the periphery; in the M1 macrophage induction group, most of the cell bodies were shrunk and the overall morphology was star-shaped, with multiple groups of short "synapse"-like structures extending from the center of the cell to the periphery; the "synapses" of the cells in the M2 macrophage induction group were thicker and elongated, and were obviously "starfish-shaped". Compared with the above three types of macrophages, the morphological characteristics and size of the macrophages in the LRG1 group were more similar to those of the M1 macrophages. Therefore, from the analysis of cell morphology, LRG1 can promote the differentiation of macrophages into the M1 type. Figure 2 It can be seen that flow cytometry detection Figure 1 The surface markers of macrophages in the four groups (blank control group, M1 macrophage induction group, M2 macrophage induction group and LRG1 stimulation group) showed that the proportion of cells positive for CD80 surface marker in LRG1 group was 82.3%, which was close to the proportion of CD80 surface marker positive cells in M1 macrophage induction group (80.1%), and the proportion of CD80 surface marker positive cells in the above two groups was much higher than that in the blank control group - 44.7% and the M2 macrophage induction group - 53.8%. The quantitative analysis of the proportion of CD80 positive cells in the four groups of macrophages also supported the significant statistical difference between the corresponding values ​​of LRG1 stimulation group and blank control group and M2 macrophage group. CD80 is a cell surface marker highly expressed by M1 macrophages, so from this flow cytometry analysis, it can be seen that LRG1 can promote the differentiation of macrophages into M1 type.

[0055] Depend on Figure 3 It can be seen that flow cytometry detection Figure 1The surface markers of macrophages in the four groups (blank control group, M1 macrophage induction group, M2 macrophage induction group and LRG1 stimulation group) showed that the proportion of cells with positive CD206 surface marker in the LRG1 group was 0.33%, which was significantly different from the proportion of cells with positive CD206 surface marker in the M2 macrophage induction group (38.6%). In the quantitative comparison of the M2 macrophage marker CD206 in the four groups of macrophages, it was found that there was a significant statistical difference in the proportion of CD206 positive cells between the LRG1 stimulation group and the M2 macrophage induction group. CD206 is a cell surface marker highly expressed by M2 macrophages. Therefore, from the flow cytometry analysis, it can be seen that the macrophages in the LRG1 stimulation group are not M2 macrophages, thus Figure 2 Together, these results further support the conclusion that LRG1 differentiates into the M1 type.

[0056] Figure 1-3 The conclusion fully proves that LRG1 can promote the differentiation of macrophages into M1 macrophages. Figure 4-5 The focus will be on explaining that macrophages stimulated by LRG1 have the tumor-killing ability of M1 macrophages. Indirect tumor-killing ability is reflected in the secretion of pro-inflammatory and anti-tumor cytoinflammatory factors by macrophages, such as tumor necrosis factor (TNFα), which exerts survival stress on tumor cells, thereby achieving the purpose of killing tumors. Direct tumor-killing ability is reflected in the direct killing of tumor cells by macrophages by adhering to tumor cells.

[0057] In addition, the relationship between LRG1 stimulation and macrophage secretion of inflammatory cytokines with anti-tumor properties was further studied, and the results are shown in Figure 4 middle.

[0058] Figure 4The protein concentration curves of six inflammatory factors with anti-tumor properties secreted by macrophages under the stimulation of different concentrations of LRG1 are shown. Among them, tumor necrosis factor (TNFα) is an inflammatory factor secreted by M1 macrophages to kill tumor cells. MCP-1, G-CSF, IL-α, IL-β and IL-6 are all pro-inflammatory factors secreted by M1 macrophages detected by ELISA, and they jointly participate in the tumor killing effect of M1 macrophages. It can be seen that the secretion of the above six anti-tumor cell behavior factors shows a significant upward trend with the increase of the concentration of LRG1 stimulation, proving that the secretion of the above six anti-tumor cell inflammatory factors and the concentration of LRG1 that stimulates macrophages show a dose-dependent upward trend. This proves that LRG1 can not only promote the differentiation of macrophages into M1 macrophages, but also promote the secretion of anti-tumor cell inflammatory factors by differentiated M1 macrophages. This directly proves that LRG1 can promote the differentiation of macrophages into M1 macrophages and promote their secretion of anti-tumor cell inflammatory factors, thereby exerting a brief tumor-killing ability on the living environment of tumor cells. Therefore, LRG1 can become a potential drug for the treatment of tumors.

[0059] In addition, the relationship between LRG1 stimulation and the adhesion ability of macrophages to tumor cells was further investigated.

[0060] The specific steps are as follows: Hela tumor cell lines were plated in 48-well plates and cultured for 24 hours. When the fusion reached 90%, 20 μg / mL ox-LDL was used to stimulate the tumor cell lines. After 24 hours of culture, the culture medium was replaced with 0.2% FBS culture medium. 100 μg LRG1 was added to the cells for intervention stimulation. Therefore, the cells were divided into four groups: 1) control group; 2) ox-LDL group; 3) LRG1 group; 4) LRG1+oxLDL group. 1×10^6 / macrophages were taken and stained with calcein-AM reagent (1 mg / mL), cultured in a 37°C incubator for 30 minutes, centrifuged at 200g for 5 minutes, resuspended in DMEM, and added to the plated tumor cells and cultured in a 30°C incubator for 1 hour. The macrophages were washed 3 times with RPMI culture medium, fixed with 4% PFA for 15 minutes, and the changes in cell number were observed with an inverted microscope (SP105F) with a 20× objective lens. The results are shown in Figure 2. Figure 5 shown.

[0061] Figure 5 The results showed that LRG1 can enhance the adhesion of macrophages to tumor cells under the stimulation of oxLDL, which is strong evidence that LRG1 promotes macrophages to directly kill tumors. M1 macrophages can sense the chemokines secreted by tumor cells under the stimulation of oxLDL and thus tend to tumor cells to enhance their adhesion to tumor cells. Figure 5It can be seen that the adhesion of macrophages to tumor cells in the LRG1 stimulation group was significantly improved, and under the stimulation of oxLDL, its adhesion was further improved, so that it adhered more and more firmly to the surface of tumor cells. In the quantitative statistics, it can be seen that the number of macrophages adhering to the surface of tumor cells in the LRG1 stimulation group was significantly higher than that of macrophages adhering to tumor cells in the blank control group, and its adhesion under oxLDL stimulation was more significantly higher than that of macrophages in the group without LRG1 stimulation. In summary, LRG1 can enhance the adhesion ability of macrophages to tumor cells, which directly proves that LRG1 can promote macrophages to directly kill tumor cells.

[0062] References

[0063] [1]Wang X,Abraham S,McKenzie JAG,Jeffs N,Swire M,Tripathi VB,LuhmannUFO,Lange CAK,Zhai Z,Arthur HM,Bainbridge J,Moss SE,Greenwood J. TGF-βsignalling.Nature.2013Jul18;499(7458):306-11.

Claims

1. Application of LRG1 in the preparation of drugs that promote the differentiation of macrophages into M1 type.

2. The use according to claim 1, characterized in that: Differentiation of macrophages to M1 type is manifested by one or more of the following: (1): enhancing the secretion of one or more inflammatory factors selected from TNFα, VCAM-1, MCP-1, IL-6, IL-1α and IL-1β, in particular TNFα and / or VCAM-1; (2): Increased expression of CD80; (3): Enhance the adhesion of macrophages to tumor cells.

3. The use according to claim 1, characterized in that: The drug is used to promote an inflammatory response against microorganisms or for anti-tumor effects.

4. The use according to claim 3, characterized in that: The tumor is a solid tumor, such as breast cancer, ovarian cancer, cervical cancer, melanoma or prostate cancer.

5. The use according to claim 3, characterized in that: The microorganisms are bacteria or viruses.

6. A macrophage activator, characterized in that The macrophage activator contains LRG1 as an active ingredient and promotes the differentiation of macrophages into the M1 type.

7. The macrophage activator according to claim 6, characterized in that The macrophage activator is used to promote inflammatory response against microorganisms or for anti-tumor effects.

8. The macrophage activator according to claim 7, characterized in that The tumor is a solid tumor, such as breast cancer, ovarian cancer, cervical cancer, melanoma or prostate cancer.

9. The macrophage activator according to claim 7, characterized in that The microorganisms are bacteria or viruses.

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