Application of LRG1 in the preparation of drugs for promoting macrophage differentiation into M1 type and macrophage activators

Abstract

The present invention relates to the use of LRG1 in the preparation of a drug for promoting macrophage differentiation into the M1 type and a macrophage activator. The present invention discovers that LRG1 activates macrophages and induces their differentiation into the pro-inflammatory M1 type, thus representing a new inducing factor for macrophage activation. This invention provides a novel mechanism for macrophage activation and offers new targets and approaches for the prevention and treatment of inflammatory responses, inflammatory-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 the M1 type and a macrophage activator. More specifically, the present invention relates to LRG1 promoting macrophage differentiation into the M1 type, thereby being used as a macrophage activator for promoting inflammatory responses against microorganisms or for anti-tumor purposes. Background Art

[0002] Macrophages participate in numerous physiological processes, including maintaining homeostasis and regulating metabolism. They also form the body's first line of defense against external danger signals. Under normal physiological conditions, macrophages function as "scavengers" within the body, playing a crucial role in maintaining tissue and organ homeostasis by stabilizing the tissue microenvironment, clearing metabolic waste, and removing damaged and diseased cells. Monocytes in the circulatory system are widely recognized as macrophage precursors. Under specific stimulation, they can differentiate into mature macrophages and travel through the circulatory system to various tissues and organs to function. Under pathological conditions, macrophages, along with other immune cells, form the body's first line of defense against external danger signals. On the one hand, macrophages can actively identify and phagocytose pathogens to prevent their spread within the body; on the other hand, macrophages are important antigen-presenting cells, directly activating the body's cellular immune response, thereby promoting a more robust immune response. In summary, macrophages play a crucial role in both innate and adaptive immunity.

[0003] Macrophages are mainly divided into two types: the M1 type that promotes inflammation and the M2 type that suppresses inflammation. Functionally, M1 macrophages can produce pro-inflammatory cytokines such as TNFα (tumor necrosis factor α), IL-1α, IL-1β, and IL-6, which inhibit the proliferation of surrounding cells. Numerous studies have shown that TNFα, IL-6, IL-1α, and IL-1β secreted by M1 macrophages can effectively enhance the macrophage's anti-tumor effect and promote the activation 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 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 primarily secrete anti-inflammatory cytokines such as IL-10, various chemokines, and growth factors to inhibit inflammatory responses, and secrete cytokines that stimulate extracellular matrix expression 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. It is secreted by the liver and is a highly conserved member of the leucine-rich repeat (LRR) family of proteins. It is primarily involved in cell adhesion, migration, and signal transduction, and is a newly discovered pro-angiogenic factor. LRG1 is expressed during granulocyte differentiation and promotes angiogenesis by inducing the conversion of transforming growth factor β (TGFbeta) signaling in endothelial cells.

[0006] Wang et al.[1] found that compared with normal mice, LRG1 expression in the retinal neovascularization model was significantly increased. 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 the upregulation of VEGF transcription and increased expression, thereby promoting endothelial cell migration and proliferation, and playing its role in 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, they found that LRG1 can promote macrophage activation. The inventors further used magnetic bead sorting to analyze changes in cytokines in the cell culture medium after LRG1 stimulation and found that LRG1 can significantly promote the secretion of inflammatory factors in macrophages. By labeling M1 and M2 macrophages with CD80 and CD206, respectively, flow cytometry analysis showed that macrophages differentiated toward the pro-inflammatory M1 phenotype after LRG1 intervention. Therefore, it was concluded that LRG1 promotes the differentiation of macrophages toward the M1 type, thus forming the present invention.

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

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

[0011] (1): Enhance 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 for promoting an inflammatory response against a microorganism or for anti-tumor use, 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 the differentiation of macrophages into the M1 type.

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

[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 number of macrophages in blood is very low, and the majority are derived from bone marrow. Bone marrow is currently widely recognized as the birthplace of macrophages. Therefore, harvesting macrophages from bone marrow is the easiest way to obtain large numbers of macrophages in experiments. Macrophages obtained using this method can generally represent the common characteristics of macrophages and serve as a universal template for macrophage research.

[0022] As used herein, in the present invention, prevention or treatment has common meaning, and should refer to the beneficial effect of the subject 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 the symptoms associated therewith; (ii) reducing the duration of the symptoms associated with the disease; (iii) preventing the progression of the disease or the symptoms associated therewith; (iv) the regression of the disease or the symptoms associated therewith; (v) preventing the development or onset of the symptoms associated with the disease; (vi) preventing the symptoms associated with the disease from reoccurring; (vii) reducing the hospitalization of the subject; (viii) reducing the length of hospitalization; (ix) the survival of the subject 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. Administered 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 discovered that LRG1 activates macrophages and induces their differentiation into the pro-inflammatory M1 type, thus representing a new inducing factor for macrophage activation. This invention provides a new mechanism for macrophage activation and offers new targets and ideas for the prevention and treatment of inflammatory responses, inflammatory-related diseases, and tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of an in vitro M1 / M2 differentiation experiment in primary macrophages are presented. Four groups were identified: a blank control group, an M1 macrophage-induced group, an M2 macrophage-induced group, and an LRG1-stimulated group. The blank control group received no added substances, while the LPS+IFNγ, IL-4, and LRG1-stimulated groups, respectively, were stimulated. The results demonstrate that LRG1 promotes the transition of macrophages to the M1 phenotype, as demonstrated by changes in BMDM morphology following stimulation in the control group, LPS+IFNγ, IL-4, and LRG1 groups.

[0025] Figure 2Flow cytometry analysis of primary macrophages induced to differentiate into the M1 type by CD80. The results indicate that LRG1 promotes the transition of macrophages to the M1 phenotype. Flow cytometry was used to identify the M1 surface marker CD80, which was used to identify the effect of LRG1 on macrophage phenotype. LRG1 stimulation of macrophages shifted the macrophage phenotype toward the M1 type.

[0026] Figure 3 Flow cytometry analysis of primary macrophages induced to differentiate into the M2 type by CD206. The results indicate that LRG1 promotes the transition of macrophages to the M1 phenotype. Flow cytometry was used to label the M2 surface marker CD206, identifying the effect of LRG1 on macrophage phenotype. LRG1 stimulation of macrophages shifted the phenotype toward the M1 type.

[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 sacrificed by cervical dislocation and disinfected by spraying 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) Remove the sterilized mouse and place it in a culture dish;

[0034] 4) Open the sterile microsurgery instrument kit, remove ophthalmic scissors and forceps, place the mouse in the supine position, and cut the skin at the hip joint along the line of the mouse's hind paw. Use forceps to peel and remove subcutaneous fat, muscle, nerves, and other tissues in sequence, fully exposing the femur and tibia and fibula. Use scissors to carefully shorten the nerve, muscle, and bone junctions. Segment the femur and tibia and fibula at the hip and ankle joints, respectively, taking care not to expose the bone marrow cavity.

[0035] 5) Take an ice box, soak the removed femur and tibia and fibula in a 15ml 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-cold PBS. Insert a 10mL syringe connected to a 1mL syringe needle into the bone marrow cavity and rapidly inject the previously drawn ice-cold PBS solution into the bone marrow cavity to flush out the bone marrow. Repeat three times to ensure sufficient bone marrow tissue is obtained from the mouse hind limb.

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

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

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

[0040] LRG1 induction interferes with macrophage polarization induction and identification of M1 and M2 phenotypes

[0041] Stimulation induction:

[0042] The macrophages cultured above 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 the figure. Figure 1 As shown, and 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 2.2 below) to confirm 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) Remove cells from the incubator after overnight intervention, remove the conditioned medium from the culture dish, wash with PBS, and then digest with trypsin for 10 minutes. Terminate the digestion with DMEM containing 100 mL / L FBS. Use a pipette to transfer the suspended cells to a 15 mL centrifuge tube and centrifuge at 500 g for 4 minutes.

[0048] 2) After centrifugation, discard the supernatant, add 1 mL of MACS solution to resuspend and wash 1-2 times, centrifuge at 500g for 4 minutes at 4°C, discard the supernatant, add 100 μL of 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: Each sample was placed in a 1.5 mL EP tube, and F4 / 80-APC and CD80-PE antibodies were added at a concentration of 1:400. The antibodies were diluted with FC-block. Single-positive tubes, F4 / 80-APC, and CD80-PE were set up, and the cells were incubated at 4°C for 30 min-1 h. Group B: Each sample was placed in a 1.5 mL EP tube, and control tubes, single-positive tubes, and test tubes were set up: blank control tube, F4 / 80-APC, CD206-BV421 tube, and F4 / 80-APC + CD206-BV421 tube. F4 / 80-APC antibody was added at a concentration of 1:400. The antibodies were diluted with FC-block. The cells were incubated at 4°C for 30 min-1 h.

[0050] 4) Antibody Washing: Centrifuge Group A, discard the supernatant, resuspend the washed antibody 1-2 times in 100-200 μL MACS buffer (PBS can be substituted), resuspend in 100 μL MACS buffer, and add DAPI (1:1000) to stain the nuclei. If storing, resuspend in PFA. Centrifuge Group B, discard the supernatant, resuspend the washed antibody 1-2 times in 100-200 μL MACS buffer (PBS can be substituted), and 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. The cells were then 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 cytometry tube and the expression of cell surface markers was detected by flow cytometry.

[0052] The experimental results show that 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 morphology of macrophages cultured in vitro was mostly plump and round, 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 shrunken 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 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 the CD80 surface marker in the LRG1 group was 82.3%, which was close to the proportion of CD80 surface marker-positive cells in the M1 macrophage induction group (80.1%). In addition, 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%). 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 the LRG1 stimulation group and the blank control group and the M2 macrophage group. CD80 is a cell surface marker highly expressed by M1 macrophages. Therefore, the flow cytometry analysis showed 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 very 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 four groups of macrophages with M2 macrophage marker CD206, 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, according to the flow cytometry analysis, the macrophages in the LRG1 stimulation group are not M2 macrophages, and 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 This study will focus on demonstrating that macrophages stimulated by LRG1 possess the tumor-killing capacity of M1 macrophages. This indirect tumor-killing capacity is manifested by the secretion of pro-inflammatory and anti-tumor cytokines, such as tumor necrosis factor (TNFα), which can coerce tumor cells into survival and ultimately kill them. Direct tumor-killing capacity, on the other hand, is manifested by macrophages directly killing tumor cells by adhering to them.

[0057] In addition, the relationship between LRG1 stimulation and the secretion of inflammatory cytokines with anti-tumor properties by macrophages 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 stimulation with 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. 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 in the concentration of LRG1 stimulation, proving that the secretion of the above six anti-tumor cell inflammatory factors shows a dose-dependent upward trend with the concentration of LRG1 that stimulates macrophages. 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 stress on the living environment of tumor cells. Therefore, LRG1 can become a potential drug for treating 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 until 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. 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). They were cultured in a 37°C incubator for 30 minutes, centrifuged at 200g for 5 minutes, and resuspended in DMEM. These macrophages were added to the plated tumor cells and cultured in a 30°C incubator for 1 hour. They were washed three times with RPMI culture medium and fixed with 4% PFA for 15 minutes. Changes in cell number were observed using 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 induced by oxLDL, which is strong evidence that LRG1 promotes macrophages to directly kill tumors. M1 macrophages can sense the chemokines secreted by tumor cells under oxLDL stimulation and thus move toward 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-stimulated group was significantly improved, and under the stimulation of oxLDL, their adhesion was further enhanced, resulting in more and more firm adhesion to the surface of tumor cells. In quantitative statistics, it can be seen that the number of macrophages adhering to the tumor cell surface in the LRG1-stimulated group was significantly higher than that of macrophages adhering to tumor cells in the blank control group. Under oxLDL stimulation, their adhesion was even 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, directly proving that LRG1 can promote the direct killing of tumor cells by macrophages.

[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 for combating cervical cancer by promoting the differentiation of macrophages into M1 type.

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

3. The use according to claim 1, characterized in that Macrophage differentiation into the M1 phenotype is manifested by enhanced secretion of TNFα and / or VCAM-1.

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