Application of substance for inhibiting or silencing activity of LAP3 in preparation of medicine for treating tumors and / or delaying immersion of listeria monocytogenes

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a substance for inhibiting or silencing LAP3 activity in preparation of a medicine for treating tumors and / or delaying immersion of listeria monocytogenes. LAP3 deletion can enhance M1 polarization of macrophages and inhibit M2 polarization at the same time. LAP3 knockout significantly inhibits polarization of tumor-associated macrophages (TAMs) to M2 type, and affects infiltration of immune cells, thereby inhibiting the progress of lung cancer and liver cancer. In addition, LAP3 knockout mice show the characteristics of delaying Listeria monocytogenes invasion and prolonging survival time. LAP3 deletion does not affect the expression level of an IFN-gamma receptor (IFNGR1 / 2), but by using an STAT6 inhibitor and a JAK1 inhibitor, M1 and M2 polarization can be recovered, and the activity of a JAK-STAT1-STAT6 signal path can be changed.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of substances that inhibit or silence the activity of LAP3 in the preparation of drugs for treating tumors and / or delaying the invasion of Listeria Background Art

[0002] Tumor is a complex disease, characterized by abnormal cell proliferation and out-of-control differentiation, ultimately forming a mass and potentially invading surrounding tissues or metastasizing to distant organs. At the molecular level, the occurrence and development of tumors involve multiple gene mutations and abnormal signaling pathways, leading to changes in key biological behaviors such as dysregulation of the cell cycle, apoptosis escape, abnormal angiogenesis, and immune escape. Although significant progress has been made in the field of current tumor treatment, many key challenges and limitations still remain. Traditional treatment methods mainly include surgical resection, radiotherapy, and chemotherapy. Although these methods are widely used clinically, each has obvious limitations. Surgical resection is only applicable to local solid tumors and has limited effect on tumors that have metastasized; radiotherapy and chemotherapy, due to their non-specific killing effects, often cause severe systemic side effects, including bone marrow suppression, immune function damage, etc.

[0003] Listeria is a Gram-positive short bacillus widely present in the natural environment. It can enter the human body through contaminated food and water, causing listeriosis. Listeriosis is a serious foodborne disease that can invade multiple organ systems, including the central nervous system, blood system, placenta, and fetus, resulting in serious consequences such as meningitis, sepsis, miscarriage, stillbirth, etc. It has a relatively high pathogenicity and fatality rate especially for high-risk groups such as pregnant women, newborns, the elderly, and immunocompromised individuals. Currently, the treatment of Listeria infection mainly relies on antibiotics such as ampicillin and penicillin. However, with the widespread use of antibiotics, the problem of Listeria drug resistance has become increasingly serious, posing a huge challenge to clinical treatment. In addition, antibiotic treatment may also cause a series of adverse reactions such as allergic reactions and intestinal flora imbalance. Therefore, finding new treatment strategies and drug targets to improve the prevention and treatment effect of Listeria infection has important clinical significance.

[0004] As an important component of the innate immune system, the polarization state of macrophages plays a key regulatory role in tumorigenesis, development, and infection immune responses. Traditional studies have shown that M1 macrophages have pro-inflammatory and anti-tumor activities, while M2 macrophages play anti-inflammatory and pro-tumor roles. The dynamic balance between the two directly affects the disease process. However, the molecular mechanisms underlying macrophage polarization regulation have not been fully elucidated, especially the lack of targets and methods that can specifically regulate the polarization direction of macrophages. In addition, the regulatory mechanisms of macrophage polarization in the tumor microenvironment are complex, and existing immunotherapy methods are difficult to achieve specific regulation of the polarization state of tumor-associated macrophages. In terms of anti-infection immunity, the polarization state of macrophages is closely related to the efficiency of pathogen clearance, but there is currently a lack of effective means to enhance anti-infection ability by regulating macrophage polarization. Therefore, developing new targets and strategies that can precisely regulate the polarization state of macrophages is of great significance for tumor immunotherapy and anti-infection therapy. Summary of the Invention

[0005] Aiming at the above deficiencies, the present invention has developed a new method for targeted regulation of macrophage polarization, which promotes macrophage polarization towards the M1 type and inhibits M2 type polarization by specifically knocking out the LAP3 gene. In tumor models, LAP3 deletion significantly inhibited the M2 polarization of tumor-associated macrophages and changed the characteristics of immune cell infiltration, thus effectively inhibiting the progression of lung cancer and liver cancer. At the same time, LAP3 knockout enhanced the anti-infection ability of the body and significantly prolonged the survival time of mice infected with Listeria monocytogenes.

[0006] The technical solution of the present invention is as follows: On the one hand, the present invention provides the use of substances that inhibit or silence LAP3 activity in the preparation of drugs for treating tumors and / or delaying the invasion of Listeria monocytogenes, and the substances achieve this by promoting the phenotypic conversion of macrophages from the M2 type to the M1 type.

[0007] Specifically, the tumors include but are not limited to: solid tumors or hematological tumors.

[0008] Preferably, the solid tumors can be lung cancer, liver cancer, breast cancer, rectal cancer, pancreatic ductal adenocarcinoma, osteosarcoma, soft tissue sarcoma, glioblastoma, ovarian cancer, or prostate cancer; The hematological tumors can be lymphoma, multiple myeloma, or leukemia.

[0009] Preferably, the tumor is lung cancer or liver cancer.

[0010] Specifically, the substances include but are not limited to: one or more of nucleic acids, Bestatin, or anti-LAP3 antibodies.

[0011] Preferably, the nucleic acid is inhibitory nucleic acid.

[0012] Preferably, the inhibitory nucleic acid is miRNA, siRNA, shRNA, antisense RNA or lncRNA; The miRNA can be complementary paired with the 3'UTR part of the target mRNA to inhibit translation or promote mRNA degradation; The siRNA binds to the RISC complex, and one of its strands (the guide strand) is completely complementary paired with the target mRNA, guiding the Argonaute protein to cleave the mRNA.

[0013] The shRNA is stably integrated into the genome through a vector (such as lentivirus) to continuously generate siRNA; The antisense RNA inhibits gene expression by direct complementary pairing. It is an important subclass of inhibitory RNA, but it does not depend on the RISC complex, and the mechanism is simpler and more direct; The lncRNA indirectly inhibits gene expression by over-recruiting chromatin modification complexes (such as PRC2) or sequestering miRNAs (ceRNA mechanism).

[0014] Specifically, the drug further comprises a pharmaceutically acceptable excipient.

[0015] Preferably, the pharmaceutically acceptable excipient is any one or more of excipients, stabilizers, diluents, binders, preservatives, lubricants, antioxidants.

[0016] Preferably, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl paraben, propyl paraben, magnesium stearate and mineral oil.

[0017] Specifically, the dosage form of the drug includes but is not limited to: tablets, liquids, capsules, powders, suppositories, granules, pills, sprays or liniments.

[0018] Specifically, the administration route of the drug is selected from oral, intravenous injection, topical, intradermal or subcutaneous injection.

[0019] The beneficial effects of the present invention are as follows: (1) Myeloid LAP3 knockout inhibits lung cancer growth and changes the proportion of immune cell infiltration in lung cancer.

[0020] (2) Myeloid LAP3 knockout promotes M2 macrophage polarization, inhibits liver cancer growth and changes the proportion of immune cell infiltration in liver cancer.

[0021] (3) Myeloid LAP3 knockout significantly inhibits the load of Listeria monocytogenes in vivo and delays the survival time of mice. Description of the Drawings

[0022] Figure 1 Knockdown of LAP3 promoted M1 polarization and inhibited M2 polarization of peritoneal macrophages. (A and B) Negative Control (NC) or siLAP3 was transfected into peritoneal macrophages (PMs) respectively, and then stimulated with IFN-γ for 12 h, and the expression of M1 macrophage markers was detected by qPCR. (C) In peritoneal macrophages with LAP3 knocked down by siRNA, after stimulation with IL-4 for 24 h, the expression of M2 macrophage markers was detected by qPCR. n = 3 (A-C). Values are expressed as mean ± SEM, **p < 0.01, ***p < 0.001, two-tailed Student's t-test.

[0023] Figure 2 Schematic diagram of the construction strategy of myeloid-specific LAP3 knockout mice and verification of LAP3 knockout efficiency. (A) Breeding scheme of Lap3fl / flLyz2-Cre+ and Lap3fl / flLyz2-Cre- mice. (B) PCR analysis of the target genes Lap3 and Lyz2-Cre transgene using the toe DNA of LAP3 mice as a template. The expression levels of Lap3 mRNA and LAP3 protein in WT and KOPMs cells were detected by qPCR and western blot. n = 3. Values are expressed as mean ± SEM, **p < 0.01, two-tailed Student's t-test.

[0024] Figure 3 Myeloid-specific knockout of LAP3 did not affect the proliferation and differentiation of macrophages. (A) Cell counting of LAP3-WT and LAP3-KOPM. (B) Detection of the expression of F4 / 80 and CD11b in LAP3-WT and LAP3-KO PM by flow cytometry. n = 3 (A-B). Values are expressed as mean ± SEM, NS indicates no statistical difference, two-tailed Student's t-test.

[0025] Figure 4 Knockout of LAP3 promoted M1 polarization and inhibited M2 polarization of bone marrow-derived macrophages. (A-D) PMs from Lap3fl / flLyz2-Cre+ (LAP3-KO-Mφ) and Lap3fl / flLyz2-Cre- (LAP3-WT-Mφ) mice were stimulated with IFN-γ / IL-4 for 12 / 24 h, and the expression of M1 / M2 macrophage markers was detected by (A and C) qPCR or (B and D) western blot. Values are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student's t-test.

[0026] Figure 5 Myeloid LAP3 knockout inhibits the growth of lung tumors in mice. (A and B) In two groups of LAP3-WT-Mφ and LAP3-KO-Mφ mice, 1×10 6 LLC cells were subcutaneously injected into each mouse. The tumor volume of the mice was measured every two days starting from the fourth day after inoculation and recorded (A). The tumor conditions and tumor weights on the 21st day after inoculation are shown in the figure (B). n = 6 (A and B), the values are expressed as mean ± SEM, **p < 0.01, two-tailed Student's t-test.

[0027] Figure 6 Myeloid LAP3 knockout promotes M1 polarization of tumor-associated macrophages (TAMs), inhibits M2 polarization, and alters the infiltration of immune cells in vivo. (A-D) Immunohistochemical staining was performed on tumor sections of LAP3-WT-Mφ and LAP3-KO-Mφ mice with the antibodies indicated as shown (A and C); and histological semi-quantification was performed (10 fields of view were selected, with 5 biological replicates in each group) (B and D). Scale bar: 100 μm. n = 10 (A and B). The values are expressed as mean ± SEM, **p < 0.01, two-tailed Student's t-test.

[0028] Figure 7 Myeloid LAP3 knockout promotes M1 polarization of tumor-associated macrophages (TAMs) and inhibits M2 polarization in vivo. (Myeloid LAP3 knockout promotes M1 polarization of TAMs in vivo). (A-B) Immunofluorescence staining was performed on tumor tissue sections of LAP3-WT-Mφ and LAP3-KO-Mφ mice with the antibodies indicated as shown (A); and histological semi-quantification was performed (10 fields of view were selected, with 5 biological replicates in each group) (B). Scale bar: 100 μm. n = 10 (A). The values are expressed as mean ± SEM, **p < 0.01, two-tailed Student's t-test.

[0029] Figure 8 Myeloid-specific LAP3 knockout inhibits the growth of liver tumors in mice. (A and B) In two groups of LAP3-WT-Mφ and LAP3-KO-Mφ mice, 5×10 6 Hepa1-6 cells were subcutaneously injected into each mouse. The tumor volume of the mice was measured every two days starting from the 4th day after inoculation and recorded, and the tumor growth curve was plotted (A). The tumor conditions and tumor weights on the 23rd day after inoculation are shown in the figure (B). n = 7 (A and B), the data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, two-tailed Student's t-test.

[0030] Figure 9Myeloid-specific LAP3 knockout promotes M1 polarization of tumor-associated macrophages (TAMs), inhibits M2 polarization, and alters the infiltration of immune cells in vivo. (A-D) Immunohistochemical staining of tumor tissue sections from LAP3-WT-Mφ and LAP3-KO-Mφ mice with the indicated antibodies; and histological semi-quantification. (A and B) Immunofluorescent staining of tumor tissue sections from LAP3-WT-Mφ and LAP3-KO-Mφ mice with the indicated antibodies; and histological semi-quantification (10 fields were selected, with 5 biological replicates in each group) (C and D). Scale bar: 100 μm. n = 10 (A and B). Values are presented as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0031] Figure 10 Myeloid LAP3 knockout alters the infiltration ratio of immune cells in lung cancer, promotes M1 polarization of TAMs, and inhibits M2 polarization. (Myeloid LAP3 knockout promotes M1 polarization of TAMs in vivo). (A) Immunofluorescent staining of tumor tissue sections from LAP3-WT-Mφ and LAP3-KO-Mφ mice with the indicated antibodies; (B) and histological semi-quantification (10 fields were selected, with 5 biological replicates in each group). Scale bar: 100μm. n = 10 (A and B). Values are presented as mean ± SEM, **p<0.01, two-tailed Student's t-test.

[0032] Figure 11 Myeloid LAP3 knockout significantly reduces the load of Listeria and delays the survival time of mice (A-E). (A and B) Listeria was administered intravenously. Survival was monitored (n = 15) (A). Three days after infection, the bacterial loads in the spleen and liver were measured (n = 5) (B). CFU, colony-forming unit. The mRNA expression levels of Nos2 and Cxcl9 in peritoneal macrophages were determined by qPCR (n = 5) (C), and the CXCL9 protein concentration in serum was measured by ELISA (n = 5) (D). T cell populations in the spleen were analyzed 7 days after infection. The left panel shows the results of a single representative experiment, and the right panel presents the cumulative data of five independent experiments (E). Scale bar: 100μm. Values are presented as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0033] Figure 12LAP3 inhibits macrophage M1 polarization and promotes macrophage M2 polarization through its enzymatic activity. (A-D) Overexpression plasmid or LAP3 enzymatic activity mutant plasmid (R368K) was co-transfected into PMs cells, and then the cells were stimulated with IFN-γ for 12 h or with IL-4 for 24 h. The expression of macrophage M1 (A) or M2 (B) marker genes was detected by qPCR. After stimulation with IFN-γ for 12 h or with IL-4 for 24 h, (C and D) the protein expression of the indicated antibodies was detected by western blot. n = 3 (A-C). Values are presented as mean ± SEM, *p < 0.05, two-tailed Student's t-test.

[0034] Figure 13 LAP3 deficiency promotes the activation of the JAK1-STAT1 signaling pathway and inhibits the activation of the STAT6 signaling pathway. (A-C) Western blot was performed using the indicated antibodies: PMs were stimulated with IFN-γ for the indicated time as shown (A); PMs were stimulated with IL-4 for the indicated time as shown (B). Values are presented as mean ± SEM, **p < 0.01, two-tailed Student's t-test.

[0035] Figure 14 LAP3 deficiency does not change the levels of IFNGR1 / IFNGR2. (A and B) Flow cytometry analysis of the changes in the levels of IFN-γ receptor IFNGR1 (A) or IFNGR2 (B) in LAP3-WT-PMs and LAP3-KO-PMs stimulated with IFN-γ at different times. n = 4 (A and B). Values are presented as mean ± SEM, **p < 0.01, two-tailed Student's t-test.

[0036] Figure 15 LAP3 promotes macrophage M2 polarization depending on STAT6 activity. (A and B) LAP3-WT-PMs and LAP3-KO-PMs were pretreated with the STAT6 inhibitor AS1517499 for 4 h and then stimulated with IL-4 for 24 h. The mRNA expression of M2-related marker genes and the protein expression of the indicated proteins were detected by qPCR (A) or western blot (B). n = 3 (A). Values are presented as mean ± SEM, **p < 0.01, two-tailed Student's t-test.

[0037] Figure 16LAP3 deficiency promotes macrophage M1 polarization depending on JAK1 activity. (A and B) LAP3-WT-PMs and LAP3-KO-PMs were pretreated with the JAK1 inhibitor Ruxolitinib for 4 h and stimulated with IFN-γ for 12 h. The mRNA expression of M1-related marker genes was detected by qPCR (A), and the corresponding protein expression was shown by western blot (B). n = 3 (A). Values are expressed as mean ± SEM, **p < 0.01, two-tailed Student's t-test. Detailed implementation mode

[0038] The present invention will be further clearly and completely described below through examples. The following examples are only a part of the examples of the present invention, which are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following examples are all conventional experiments unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0039] Example 1 Role of leucine aminopeptidase 3 (LAP3) in macrophage polarization 1.1 Experimental method 1.1.1 Construction of myeloid-specific LAP3 knockout mice and identification of mouse genotypes (1) Construction of myeloid-specific LAP3 knockout mice Myeloid-specific LAP3 gene knockout mice Lap3 fl / fl (C57BL / 6J) mice were purchased from Cyagen Biosciences Inc., Guangzhou, China and bred using CRISPR-Cas9-mediated genome editing technology. Myeloid knockout tool mice Lyz2 -Cre (C57BL / 6J) was provided by the team of Professor Tan Xiaoyue at Nankai University. It was crossed with Lap3 fl / fl mice to obtain myeloid cell-specific LAP3 gene knockout mice Lap3 fl / fl Lyz2- Cre + / - , and the littermate-bred Lap3 fl / fl Lyz2- Cre - / -Mice were used as experimental control mice. All mice were housed under specific pathogen-free (SPF) conditions in the Department of Laboratory Animal Science, Tianjin Medical University. The environmental temperature was maintained at 22 - 24 °C, and free access to food and water was provided, with a 12-hour light-dark cycle. Male and female mice were randomly divided into an experimental group and a littermate control group. During the experiment, the requirements of the "Guidelines for the Ethical Review of Laboratory Animal Welfare (GB / T 35892 - 2018)" were strictly followed, and the welfare ethics of laboratory animals were implemented in a standardized manner. All experiments have been approved by the Institutional Animal Care and Use Committee (IACUC) of Tianjin Medical University.

[0040] 1.1.2 Extraction and culture of primary mouse cells Extraction and culture of peritoneal macrophages (PMs): (1) Healthy mice aged 8 - 12 weeks were selected. Three days before the experiment, 1 mL of 3% thioglycollate solution was injected intraperitoneally into each mouse.

[0041] (2) Three days later, the mice were sacrificed by cervical dislocation, soaked in 75% ethanol for 3 - 5 minutes for disinfection, wiped with absorbent paper to clean the surface, and then fixed supine on the dissection board and placed in a laminar flow hood for subsequent operations.

[0042] (3) Hold the syringe, gently insert the needle of a 1 mL syringe into the subcutaneous tissue in the right lower abdomen with the cooperation of a 10 mL syringe. After entering the abdominal cavity, slowly inject 8 mL of pre-cooled 3% FBS solution, and then gently massage the abdomen for 5 minutes (note not to puncture the abdominal organs and intestinal cavity, otherwise the syringe should be discarded and the mouse should be replaced to avoid cross-infection).

[0043] (4) Use a 5 mL syringe to aspirate the peritoneal lavage fluid and transfer it to a 10 mL centrifuge tube. Lift the abdominal skin with sterile hemostatic forceps and make a small incision about 1 cm long in the abdomen with sterile scissors. After keeping the hemostatic forceps lifted, use a 1 mL pipette to penetrate the abdominal cavity from the incision to aspirate the remaining peritoneal lavage fluid. Aspirate 1 mL of 3% FBS solution and rinse the abdominal cavity twice repeatedly.

[0044] (5) Centrifuge the collected lavage fluid at 1000 rpm for 5 minutes at room temperature. If there is bleeding in the abdominal cavity, 2 mL of red blood cell lysis buffer can be added after centrifugation, and left to stand at room temperature for 5 minutes to lyse the red blood cells. After filtering through a 70 μm filter, centrifuge again and remove the lysis buffer. Then, resuspend the cells in 1×PBS buffer, centrifuge at 1000 rpm for 5 minutes at room temperature, remove the supernatant, and resuspend the cell pellet in an appropriate amount of complete RPMI-1640 medium. Gently pipette to disperse the cells evenly. Plate the resuspended cells in a 10 cm diameter cell culture dish or a 12-well plate, and culture them in an incubator with 5% CO2 at 37°C. After standing for 2 - 3 hours, wash away the non-attached cells with complete medium, and the remaining adherent cells are peritoneal-derived macrophages.

[0045] 1.1.3 Detection of the effects of LAP3 deficiency on the proliferation and differentiation of mouse macrophages by flow cytometry To study the effects of myeloid-specific LAP3 knockout on the proliferation and differentiation of mouse macrophages, flow cytometry was used to detect the expression ratios of the markers CD11b and F4 / 80 in peritoneal macrophages (PMs) from WT-Mφ and KO-Mφ mice. The specific experimental steps are as follows: (1) Select healthy mice aged 8 - 12 weeks. Three days before the experiment, inject 1 mL of 3% thioglycollate solution into the abdominal cavity of each mouse.

[0046] (2) Three days later, euthanize the mice by cervical dislocation, soak the mice in 75% ethanol for 3 - 5 minutes for disinfection, wipe the surface with absorbent paper for cleaning, then fix the mice supine on the dissection board and place them in a laminar flow hood for subsequent operations.

[0047] (3) Hold the syringe, gently insert the needle of a 1 mL syringe into the subcutaneous tissue of the right lower abdomen with the cooperation of a 10 mL syringe. After entering the abdominal cavity, slowly inject 8 mL of pre-cooled 3% FBS solution, and then gently massage the abdomen for 5 minutes (note not to puncture the abdominal organs and intestinal cavity, otherwise the syringe should be discarded and the mouse replaced to avoid cross-infection).

[0048] (4) Use a 5 mL syringe to aspirate the lavage fluid in the abdominal cavity and transfer it to a 10 mL centrifuge tube. Lift the abdominal skin with sterile hemostatic forceps and make a small incision about 1 cm long in the abdomen with a sterile scissors. While keeping the hemostatic forceps lifted, use a 1 mL pipette to reach into the abdominal cavity through the incision to aspirate the remaining abdominal lavage fluid. Aspirate 1 mL of 3% FBS solution and rinse the abdominal cavity repeatedly 2 times.

[0049] (5) Centrifuge the collected lavage fluid at 1000 rpm for 5 min at room temperature. If there is abdominal bleeding, 2 mL of red blood cell lysate can be added after centrifugation, and the mixture is allowed to stand at room temperature for 5 min to lyse the red blood cells. After filtration through a 70 μm filter, centrifuge again and remove the lysate. Then, resuspend the cells twice with 1×PBS buffer and wash, and centrifuge at 1000 rpm for 5 min at room temperature.

[0050] (6) Discard the supernatant, flick the bottom of the centrifuge tube, and resuspend the cells with 1×PBS buffer. After pipetting and mixing evenly, take an appropriate amount of cell suspension for viable cell counting and dispense it into 1.5 mL EP tubes (sample tube, unstain tube, single stain tube).

[0051] (7) Centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, and add 50 μL of CD16 / CD32 to block the Fc segment (0.25 μg / 100 μL, prepared with 3% BSA), and incubate on ice for 30 min.

[0052] (8) Add 10 μL of directly labeled primary antibody diluent CD11b-APC, F4 / 80-PE-Cy5 (6:200, prepared with 3% BSA) to each tube, and incubate in the dark on ice for 1 hour.

[0053] (9) Wash the cells twice with 3% BSA, each time centrifuge at 1000 rpm and 4°C for 5 min.

[0054] (10) Finally, resuspend the cells with 200 μL of 1×PBS buffer, filter the suspension into a flow cytometry tube, store in the dark on ice, and prepare for loading onto the machine.

[0055] (11) Use FlowJo software for data analysis.

[0056] Table 1 Flow cytometry antibodies

[0057] 1.1.4 Tissue immunofluorescence (1) Tissue frozen section: The section thickness is 5 μm, stored at -20°C, taken out and placed at room temperature for 5 min before staining to ensure that the tissue adheres firmly to the glass slide.

[0058] (2) Fixation and permeabilization: Pre-cool anhydrous methanol in a -20°C refrigerator for 20 min, immerse the thawed frozen section in anhydrous methanol, and fix in a -20°C refrigerator for 15 min.

[0059] (3)Blocking: Wash the sections twice with TBST (80 rpm for 5 min each time), then block with 3% BSA (prepared with TBST) and incubate at room temperature for 1 h. After blocking, wash twice again with TBST (80 rpm for 5 min each time).

[0060] (4)Incubating with primary antibody: Dilute the primary antibody according to the ratio recommended in the instruction manual, prepare the primary antibody solution with the blocking solution, drop it onto the tissue surface, then place the sections in a wet box and incubate overnight at 4°C. Wet tissue paper should be placed in the wet box to maintain humidity. The tissue around the sections can be circled with a histochemical pen in advance to ensure that the tissue can be soaked in the primary antibody solution.

[0061] (5)Incubating with secondary antibody: Wash the sections three times with TBST (80 rpm for 10 min each time), prepare the secondary antibody solution with the blocking solution according to the antibody species information. Then incubate at room temperature in the dark for 1 h (the incubation time can be appropriately extended).

[0062] (6)DAPI staining: Wash the sections three times with TBST (80 rpm for 10 min each time), then stain the cell nuclei with DAPI diluent and incubate at room temperature in the dark for 10 min (the dilution ratio of DAPI is 1:1000).

[0063] (7)Cover slipping: Wash the sections three times with TBST (80 rpm for 10 min each time), and cover slip with a fluorescence mounting medium. After cover slipping, cover with aluminum foil and let it stand at room temperature until the cover glass is fixed, then store the sections in a 4°C refrigerator.

[0064] (8)Microscopic observation and image analysis: Observe the sections with a fluorescence microscope or a confocal microscope and capture images, and perform image analysis using ImageJ or Photoshop software. The immunofluorescent antibodies used in this experiment are shown in Table 2.

[0065] Table 2 Immunofluorescent antibodies

[0066] 1.1.5 Establishment of tumor-bearing mouse model (taking liver cancer as an example) (1)Select 15 WT-Mφ and 15 KO-Mφ mice at 8 - 12 weeks old, and subcutaneously inject mouse liver cancer cells Hepa1-6 (5×10 6 / mouse). When inoculating cells, cells in good growth condition, free of virus, bacteria or mycoplasma contamination, and in the logarithmic growth phase should generally be selected to establish a hepatocellular carcinoma tumor model.

[0067] (2)Observe the tumor growth, record the tumor volume, and draw a tumor growth curve. When the tumor grows for about 28 days, sacrifice the mice.

[0068] (3) Collect mouse tumor tissues, embed the tumor tissues and section them, and detect the infiltration of immune cells in the tumor tissues by immunohistochemical staining, such as the expression of myeloid cells positive for CD11b, T cells positive for CD4 and CD8, and regulatory T cells positive for Foxp3. Detect the types and infiltration ratios of macrophages in the tumor tissues by immunohistochemistry and fluorescence staining.

[0069] 1.1.6 Listeria monocytogenes-infected mouse model Thaw the frozen single-strain bacilli. Eight-week-old mice were injected via the marginal tail artery with a lethal (2.3×10 5 CFUs) or sub-lethal (5×10 4 CFUs) bacterial dose, which was suspended in 200 μL of PBS. On day 3 post-infection, the spleen and liver were collected and lysed in PBS containing 0.05% Triton X-100, and bacterial CFUs were determined by plating on BHI plates. Total RNA was extracted from peritoneal macrophages to determine the expression of classical M(IFN-γ) signature genes. Blood was collected from the inferior vena cava and serum CXCL9 concentration was measured using ELISA. On day 7 post-infection, CXCR3+ T cell populations in the spleen were analyzed by flow cytometry.

[0070] 1.1.7 Statistical analysis methods All data statistics and analysis in this section were performed using GraphPad Prism software, which was also used for chart drawing. To ensure the accuracy and reliability of experimental data, all quantitative experimental data included three biological replicates and three technical replicates, and the differences between samples were shown in the charts. Data were expressed as mean ± SEM, and differential analysis was performed using two-tailed Student's t-test. One-way analysis of variance (ANOVA) was used for comparison of multiple groups of data, and log-rank test (Mantel-Cox) was used for comparison of survival curves. The criteria for determining statistical significance were: NS (p ≥ 0.05) indicating no statistical difference, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 indicating statistical significance.

[0071] 1.2 Results 1.2.1 LAP3 inhibits M(IFN-γ) polarization and promotes M(IL-4) polarization To investigate the effect of LAP3 on macrophage polarization, LAP3 was knocked down by small interfering RNA (siRNA) in peritoneal macrophages (PMs) Figure 1in A); at the same time, the cells were stimulated with IFN-γ for 12 h, and it was detected by fluorescence quantitative real-time PCR technology that after knocking down LAP3 compared with the control group, the mRNAs expression of M1-type specific marker genes such as Il-6, Nos2, Il-1β, and Tnf-α was significantly increased. Correspondingly, in PM cells with LAP3 knocked down by siRNA, they were stimulated with IL-4 for 24 h, and it was found that the mRNAs expression of M2-type specific marker genes such as Arg1, Retnla, Mgl1, and Mgl2 decreased after LAP3 was knocked down ( Figure 1 in B and C). The above results indicate that knocking down LAP3 at the gene level promotes the polarization of peritoneal macrophages to M(IFN-γ) and inhibits the polarization to M(IL-4).

[0072] 1.2.2 Construction and verification of myeloid LAP3-specific knockout mice To further explore the effect of LAP3 on macrophage polarization, mice with myeloid-specific knockout of the LAP3 gene were constructed Lap3 fl / fl Lyz2 -Cre + (LAP3-KO-Mφ) and Lap3 fl / fl Lyz2 -Cre - (LAP3-WT-Mφ) were used to restrict the production of leucine aminopeptidase 3 so as to explore its effect on macrophage polarization. The Lyz-Cre tool mouse is a genetically engineered mouse model commonly used to study the functions of specific genes. In this model, Cre recombinase is specifically expressed in myeloid cells such as macrophages and neutrophils. The Lap3 + / + Lyz2 -Cre + mice were mated with Lap3 + / + Lyz2 -Cre - mice, and the obtained offspring Lap3 fl / + Lyz2 -Cre + mice were mated with Lap3 + / + Lyz2 -Cre - to obtain mice with myeloid-specific knockout of Lap3 Lap3 fl / fl Lyz2 -Cre + (LAP3-KO-Mφ) and its control mice Lap3 fl / fl Lyz2 --Cre - (LAP3-WT-Mφ) ( Figure 2 A in). After amplification and reproduction of the mice, the genotypes of the mice were identified by PCR, and then peritoneal macrophages PMs of WT and KO mice were extracted. RT-qPCR and western blot methods were used to detect the knockdown efficiency of LAP3. The results showed that the gene and protein expression levels of LAP3 in primary macrophages of LAP3-KO mice were significantly decreased ( Figure 2 B in).

[0073] Next, the effects of myeloid-specific knockout of Lap3 on macrophage proliferation and differentiation were explored. After extracting peritoneal macrophages, cell counting and flow cytometry were used to analyze the markers F4 / 80 and CD11b representing macrophages. The results showed that there were no significant changes in the total number of peritoneal macrophages, the number of F4 / 80-positive and CD11b-positive macrophages, and the proportion of F4 / 80 and CD11b double-positive macrophages in the Lap3-KO group compared with the WT group ( Figure 3 A-B in), indicating that myeloid-specific knockout of Lap3 had no effect on the proliferation and differentiation of mouse macrophages. The above results showed that the mouse model with myeloid-specific knockout of Lap3 was successfully constructed and did not affect the proliferation and differentiation of macrophages.

[0074] 1.2.3 Knockout of LAP3 in PM promotes macrophage M1 polarization and promotes M2 polarization PM cells of myeloid LAP3 knockout mice were extracted and stimulated with IFN-γ or IL-4 at the same time. It was detected by fluorescence quantitative real-time PCR technology and western blot that after myeloid LAP3 knockout, compared with the wild-type group, Il-6 , Nos2 , Il-1β and Tnf-α the mRNAs expression of these M1-type specific marker genes was significantly increased and the protein expression of iNOS was enhanced. Correspondingly, after stimulating PM cells with IL-4 for 24 h after myeloid LAP3 knockout, it was found that the mRNAs expression of these M2-type specific marker genes such as Arg1, Retnla, Mgl1, and Mgl2 was significantly decreased and the expression of ARG1 was inhibited after myeloid LAP3 knockout ( Figure 4 A-D in).

[0075] 1.2.4 Myeloid LAP3 knockout inhibits lung cancer growth and changes the proportion of lung cancer immune cell infiltration Next, to further investigate the role of myeloid LAP3 knockout in macrophage polarization in vivo, relevant in vivo experiments were conducted, and a basic model related to induced polarization was established using myeloid LAP3 knockout mice. Mouse Lewis lung carcinoma (LLC) cells were selected for tumor-bearing experiments, and LLC cells (1×10 6 were subcutaneously inoculated into mice with LAP3-WT-Mφ and LAP3-KO-Mφ. Starting from the fourth day after inoculation, the tumor volume of the mice was measured every 2 days, and the mice were sacrificed on the 21st day after inoculation, and the tumor tissues were dissected for photography and weighing. It was found that myeloid LAP3 knockout significantly inhibited the growth of mouse tumors ( Figure 5 A and B in).

[0076] Next, the tumor tissues were sectioned, and then the infiltration of immune cells in the tumor cells was detected by immunohistochemical staining. The results showed that, compared with LAP3-WT-Mφ mice, in the tumor tissue sections of LAP3-KO-Mφ mice, the number of CD11b-positive cells (myeloid cells) and CD4-positive cells (CD4 T + cells) was almost twice that of LAP3-WT-Mφ mice, while the number of CD8α-positive cells (CD8 T + cells) increased by about 3 times. The staining results of the regulatory T cell (Treg) specific marker FoxP3 showed that the number of regulatory T cells decreased to about 50% ( Figure 6 A and C in). Subsequently, the infiltration and phenotype of tumor-associated macrophages (TAMs) in the tumor tissue sections were explored. The results showed that the number of F4 / 80-positive macrophages increased by 2 times, the expression of the M1-related marker gene iNOS was significantly upregulated, while the expression of the M2-related marker gene ARG1 was significantly downregulated ( Figure 6 B and D in). These results indicate that myeloid LAP3 knockout changes the infiltration of immune cells in tumor tissues and promotes the polarization of TAMs to M1.

[0077] In addition, immunofluorescence staining was also performed on the tumor tissue sections of LAP3-WT-Mφ and LAP3-KO-Mφ mice. Consistent with the previous immunohistochemical results, the immunofluorescence staining results showed that the number of TAMs expressing iNOS increased significantly while the number of TAMs expressing ARG1 decreased significantly ( Figure 7 A and B in). Combining the results of immunofluorescence and immunohistochemistry indicates that myeloid LAP3 knockout promotes the M1 polarization of TAMs and inhibits the M2 polarization.

[0078] 1.2.5 Myeloid LAP3 knockout promotes M2 macrophage polarization, inhibits liver cancer growth and changes the proportion of immune cells infiltrating liver cancer The role of promoting M2 macrophage polarization after LAP3 knockout in cancer development was further investigated using a tumor-bearing model. First, a basic model related to induced polarization was established using mice with myeloid LAP3 knockout. Mouse Hep1-6 liver cancer cells were selected for the tumor-bearing experiment. LLC cells (1×10 6 ) were subcutaneously inoculated into mice with LAP3-WT-Mφ and LAP3-KO-Mφ. From the 4th day after inoculation, the tumor volume of the mice was measured every 2 days, and the curve was plotted according to the tumor growth time. The mice were sacrificed on the 23rd day after inoculation, and the tumor tissues were dissected, photographed and weighed (A and B in Figure 8 ). The results showed that the tumor volume and weight of LAP3 knockout mice were significantly reduced, indicating that myeloid LAP3 knockout significantly inhibited the growth of mouse tumors.

[0079] Next, the collected tumor tissues were embedded and sectioned, and then immunohistochemical staining was used to detect the infiltration of immune cells in the tumor tissues. The results showed that, compared with LAP3-WT-Mφ mice, in the tumor tissue sections of LAP3-KO-Mφ mice, the number of CD11b-positive cells (myeloid cells), CD4-positive cells (CD4 + T cells) and CD8a-positive cells (CD8 + T cells) was almost twice that of LAP3-WT-Mφ mice; while the staining result of the specific marker FoxP3 of regulatory T cells (Treg cells) showed that the number of regulatory T cells decreased to about 50% (A and B in Figure 9 ). Then, the infiltration and polarization phenotypes of TAMs in the tumor tissue sections were explored. The results showed that the number of F4 / 80-positive macrophages increased by about 2 times, and the expression level of the M1-related marker gene iNOS was significantly up-regulated, while the expression level of the M2-related marker gene ARG1 was significantly down-regulated (C and D in Figure 9 ). These results above indicate that myeloid-specific knockout of LAP3 can change the infiltration of immune cells in tumor tissues, promote the formation of an anti-tumor immune microenvironment, and promote the polarization of TAMs into the M1 type and inhibit their polarization into the M2 type.

[0080] In addition, immunofluorescence staining was also performed on the tumor tissue sections of LAP3-WT-Mφ and LAP3-KO-Mφ mice. Consistent with the previous immunohistochemical results, the immunofluorescence staining results showed that the number of TAMs expressing iNOS increased significantly while the number of TAMs expressing ARG1 decreased significantly ( Figure 10A and B in). The results of combining immunofluorescence and immunohistochemistry showed that myeloid LAP3 knockout promoted M1 polarization of TAMs and inhibited M2 polarization.

[0081] 1.2.6 Myeloid LAP3 knockout significantly inhibited the load of Listeria in vivo and prolonged the survival time of mice Next, the role of LAP3 in the in vivo immune response was investigated. After injecting LAP3-WT-Mφ and LAP3-KO-Mφ mice with a lethal dose of Listeria monocytogenes, the survival rate of LAP3-KO-Mφ mice was significantly increased ( Figure 11 A in) and the bacterial load in the spleen and liver was reduced ( Figure 11 B in). Peritoneal macrophages were extracted from LAP3-WT-Mφ and LAP3-KO-Mφ mice. qPCR analysis showed that the expression of Nos2 and Cxcl9 in the LAP3 knockout group was significantly enhanced compared with the WT group ( Figure 11 C in). Consistent with this, the concentration of CXCL9 protein in the serum increased ( Figure 11 D in). Since CXCL9 is an important chemokine for recruiting CXCR3 + T cells, LAP3-KO-Mφ mice showed enhanced recruitment of CXCR3 + T cells to the spleen after infection ( Figure 11 E in). Therefore, LAP3 knockout enhanced the immune effector function of macrophages in vivo and strengthened the host defense.

[0082] 1.2.7 The regulatory effect of LAP3 on macrophage polarization does not depend on its enzymatic activity To further explore whether the regulation of macrophage polarization by LAP3 depends on its enzymatic activity, WT-LAP3 and the enzymatically inactive mutant LAP3-R368K plasmids were overexpressed in LAP3-WT-PM and LAP3-KO-PM, and at the same time, they were stimulated with IFN-γ or IL-4. The results showed that LAP3-KO-PM would increase the expression of macrophage M1 marker genes, such as Il-1β , Il-6 and Nos2 ; when the WT-RFK plasmid was overexpressed simultaneously, the expression of these M1 marker genes did not increase, but when the enzymatically inactive mutant RFK plasmid was overexpressed, the expression of these M1 marker genes still increased ( Figure 12 A and B in). Correspondingly, it was also found that LAP3-KO-PM would inhibit the expression of macrophage M2 marker genes, such as Arg1 , Retnla and Mgl2; When overexpressing the WT-LAP3 plasmid simultaneously, the expression of these M2 marker genes will not increase. However, when overexpressing the enzymatically inactive mutant LAP3-R368K plasmid simultaneously, the expression of these M2 marker genes will increase ( Figure 12 C and D in

[0083] ). The above results indicate that the effect of LAP3 on macrophage polarization depends on its enzyme activity.

[0084] In summary, knocking out LAP3 promotes macrophage M(IFN-γ) and inhibits M(IL-4) polarization. By knocking down LAP3 using siRNA at the gene level or constructing myeloid LAP3 knockout mice, after stimulating macrophages with IFN-γ or IL-4, through detection methods such as qPCR and western blot, the experimental results show that LAP3 inhibits macrophage M(IFN-γ) and promotes M(IL-4) polarization. Then, a plasmid LAP3-R368K with an enzymatically inactive mutant of LAP3 was constructed to verify that LAP3 regulates macrophage polarization depending on its enzyme activity.

[0085] Example 2 Study on the mechanism of action of leucine aminopeptidase 3 in regulating macrophage polarization 2.1 Experimental methods 2.1.1 Detection of the effect of LAP3 deficiency on the expression of IFNGR1 / 2 on the surface of mouse macrophages by flow cytometry The methods used in this part of the experiment have been described in the first part. For details, see 1.1.3. The flow cytometry antibodies involved in this part are shown in the following table.

[0086] Table 3 Flow cytometry antibodies

[0087] 2.1.2 Protein extraction and western blot assay (WB) (1) Extraction of cellular proteins (taking a 12-well plate as an example) Observe the cells under a microscope, select the culture dishes with good cell growth status under the microscope, rinse the cells twice with 1×PBS buffer, discard the PBS buffer, then add 200 μL of 1×SDS to each well, place it on ice for 5 min, scrape the cells with a clean cell scraper, collect the cells with a pipette and put them into a 1.5 ml EP tube, perform denaturation treatment at 100 °C in a metal bath for 10 min, place it in a pre-cooled centrifuge at 4 °C for a short centrifugation, take it out and make marks, and store it in a -80 °C refrigerator for long-term preservation.

[0088] (2)Preparation of SDS-PAGE electrophoresis gel Use two clean glass plates. After aligning the glass plates, place them in a clamp and tighten, and fix them on the gel-making support. According to the molecular weight of the target protein to be separated, refer to the following table to prepare the separation gel with the optimal concentration. Add the required volumes of reagents in sequence from left to right according to the reagent order in the table, gently stir and mix evenly, quickly inject the separation gel into the gap between the two glass plates, leaving space for pouring the stacking gel. After adding the separation gel, carefully drip isopropanol with a dropper to press the gel. After it solidifies, discard the isopropanol, and then wash the top of the gel several times with ddH2O. After the washing is completed, try to remove the residual water on the upper layer of the gel as much as possible. Prepare the required volume of stacking gel according to the following table, and then directly pour the stacking gel on top of the separation gel. Insert a 10- or 15-well comb according to the experimental purpose to form sample loading wells, and let it stand until it solidifies. Wrap it with a clean and moist paper towel and store it at 4 °C for later use.

[0089] Table 4 Separation gel preparation formula

[0090] Table 5 Stacking gel formula

[0091] (3)Electrophoresis Fill the electrophoresis tank with 1×Running buffer until it overflows to the outer tank. If there are bubbles in the sample loading wells, use the electrophoresis buffer to blow and remove the bubbles in the sample loading wells. Add 5 μL of protein Marker to the sample loading wells, and then sequentially add the protein samples to be electrophoresed at a uniform speed. If there are extra wells, supplement the loading buffer on both sides of the sample loading wells to flatten them. Different groups are indicated and separated by the corresponding protein maker. After loading the samples, turn on the power supply and perform electrophoresis at a constant voltage. First, electrophorese at a constant voltage of 80 V for about 30 min. When the protein is flattened in the separation gel, use a constant voltage of 120 V for electrophoresis until the indicator is about 1 cm away from the bottom end of the gel. Once the target protein is properly separated, the electrophoresis can be stopped.

[0092] (4)Transfer Cut a PVDF membrane of appropriate size according to the gel and write the corresponding gel plate and protein label. Then immerse the PVDF membrane in anhydrous methanol for activation for about 30 s. Pour an appropriate amount of 1×Transfer buffer into the tray, place the sponge pad into the 1×Transfer buffer, and then lay three pieces of filter paper slightly larger than the PVDF membrane flat on the sponge pad. Cut off the stacking gel in the gel plate and keep the separating gel. Place the components in the order of the negative end of the transfer membrane clamp, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and positive end according to the "sandwich model", and carefully clamp them (keep no bubbles on the gel surface). Place the assembled fixture into the electrophoresis tank according to the corresponding electrode, pour an appropriate amount of 1×Transfer buffer, place an ice box and cover it with ice, and perform membrane transfer at a constant voltage of 90 V for a time generally of 1 - 2 h. (Adjust the membrane transfer time and voltage according to the size of the target band.) (5)Blocking After the membrane transfer is completed, take out the PVDF membrane, wash it three times with 1×TBST, and then place the PVDF membrane face up in a plastic dish containing 5% skim milk, and place it on a shaker at room temperature for slow blocking at a speed of 20 rpm for 1 h.

[0093] (6)Primary antibody and secondary antibody incubation After blocking is completed, wash the PVDF membrane 3 times with 1×TBST, 5 min each time. Then cut the PVDF membrane according to the molecular weight of the target protein, and then place the PVDF membrane in an incubation box and add the corresponding primary antibody to completely cover it (the dilution multiple can be referred to the antibody instruction manual and diluted to an appropriate concentration with the primary antibody dilution solution), place it in the refrigerator shaker for overnight incubation at a temperature of 4 °C. After the incubation is completed, recover the primary antibody, place it on the shaker at room temperature and wash the membrane three times with 1×TBST, with a washing time of 10 min each time (the rotation speed is 100 rpm). After washing the membrane, incubate the secondary antibody corresponding to the primary antibody (diluted with 5% skim milk, and the dilution multiple can be referred to the antibody instruction manual), and incubate at low speed at room temperature for 1 h (20 rpm).

[0094] (7)Secondary antibody incubation After the primary antibody incubation is completed, wash the PVDF membrane 3 times with 1×TBST on a shaker at a rotation speed of 80 rpm, 5 min each time. Prepare the secondary antibody (1:5000) with 5% skim milk (prepared with TBST) according to the species attribute of the primary antibody, and block it at low speed (20 rpm) on a shaker at room temperature for 1 h.

[0095] (8)Developing and exposing After the primary antibody incubation was completed, the membrane was washed 3 times with 1×TBST on a shaker at 80 rpm for 5 min each time. Prepare the developing solution (Solution A + Solution B = 1:1, stored in the dark and freshly prepared before use). Use forceps to pick up the target band and place it in a glass dish. Sufficiently add the developing solution to ensure that the developing solution evenly infiltrates all parts of the membrane. Place the membrane in the machine for exposure imaging.

[0096] 2.2 Results 2.2.1 Leucine aminopeptidase 3 can inhibit the activation of the JAK1-STAT1 signaling pathway and promote the activation of the STAT6 signaling pathway Next, explore how LAP3 regulates macrophage polarization. It is known that the activation of STAT1 plays a key role in macrophage M1 polarization, while the activation of STAT6 is required for the activation of M2 macrophages. First, WT-LAP3 and KO-LAP3 cells were stimulated with IFN-γ for 0, 15, and 30 minutes to investigate the changes in the JAK1-STAT1 signaling pathway. Compared with the control group, PMs with myeloid LAP3 knockout and systemic LAP3 knockout showed enhanced activation of STAT1 and its upstream JAK1 and JAK2 under IFN-γ stimulation ( Figure 13 A and B in). The above results indicate that primary macrophage LAP3 inhibits the activation of IFN-γ-induced STAT1 and its upstream kinases JAK1 and JAK2 ( Figure 13 B in). Next, explore the mechanism by which LAP3 promotes macrophage M2 polarization. Therefore, the STAT6 signaling pathway, which classically activates macrophage M2 polarization, was detected. First, mouse PMs were stimulated with IL-4 for 0, 15, and 30 minutes, and consistent results were also obtained, that is, it was found that compared with the cells in the WT group, the deletion of myeloid LAP3 inhibited the effect of IL4-induced STAT6 activation on the macrophage polarization-related signaling pathway ( Figure 13 C in). These above results indicate that the deletion of LAP3 can inhibit the activation of the STAT6 signaling pathway.

[0097] 2.2.2 Deletion of LAP3 does not change the expression of IFN-γ membrane receptors IFN-γRα and IFN-γRβ As the receptor of IFN-γ, IFNGR1 and IFNGR2 activate the JAK1 / 2 signal after contacting the signal of ligand IFN-γ, and then phosphorylate downstream STAT1 to regulate macrophage polarization. It was explored that STAT1 could be activated and the phosphorylation of its upstream JAK1 and JAK2 could be induced under IFN-γ induction after LAP3 knockout. Next, in order to further explore the mechanism of LAP3 regulating macrophage polarization, the expression levels of the receptors of IFN-γ, IFN-γRα and IFN-γRβ were detected. WT and KOPMs were stimulated with IFN-γ and analyzed by flow cytometry. The results showed that the knockout of LAP3 did not induce the up-regulation of the expression of IFN-γ receptor subunits IFNGR1 and IFNGR2 compared with the control group under the basal level or the condition stimulated by IFN-γ ( Figure 14 A and B in). It indicates that the enhanced activation of the JAK1-STAT1 signaling pathway caused by LAP3 is not due to the change in the level of IFN-γ receptor.

[0098] 2.2.3 LAP3 promotes macrophage M2 polarization depending on STAT6 activity LAP3 promotes the M2 polarization of macrophages by activating STAT6, which is based on the key role of STAT6 in the polarization of M2 macrophages. M2 macrophages usually have anti-inflammatory and tissue repair-promoting functions. When cells were pretreated with the STAT6 inhibitor AS1517499, the activity of STAT6 could be inhibited, thus restoring the expression of M2 marker genes after LAP3 knockout. Through experimental data, LAP3 promotes the M2 polarization of macrophages by activating STAT6 ( Figure 15 A and B in). As a key transcription factor, STAT6 plays an important role in this process. The results also showed that the use of the STAT6 inhibitor AS1517499 could effectively reverse the decrease in the expression of M2 macrophage markers caused by LAP3 knockout, which further supported the close relationship between LAP3 and the STAT6 pathway.

[0099] 2.2.4 Lack of LAP3 inhibits macrophage M2 polarization depending on JAK1 activity Regarding the role of the JAK-STAT1 pathway in the polarization of LAP3-deficient macrophages, the experimental results showed that pretreatment of cells with the JAK1 inhibitor Ruxolitinib before IL-4 stimulation could reverse the phenomenon of decreased expression of macrophage M2-related marker genes caused by LAP3 deficiency ( Figure 16A and B in). This indicates that the JAK1 pathway may play a key role in the M2 polarization of macrophages lacking LAP3. The activation of the JAK-STAT1 pathway is usually associated with the polarization of M1 macrophages, while in the polarization of M2 macrophages, the role of JAK1 may be to regulate the expression of related genes, thereby affecting the immune response of macrophages. Through the intervention of the JAK1 inhibitor Ruxolitinib, it was experimentally observed that the expression of M2-related genes was restored, which provided direct evidence for understanding the role of the JAK-STAT1 pathway in macrophage dysfunction under LAP3 deficiency. In summary, the JAK1 inhibitor Ruxolitinib can reverse the abnormal macrophage function caused by LAP3 deficiency by intervening in the JAK1 / STAT pathway, suggesting that the JAK-STAT1 pathway may be one of the key regulatory factors of LAP3 in macrophage polarization.

[0100] 2.2.5 LAP3 deficiency promotes M1 polarization of macrophages dependent on JAK1 activity In the above experiments, the role of the JAK-STAT signaling pathway in the polarization of M2 macrophages was explored. Next, its role in M1 macrophages was studied. Therefore, by pretreating cells with the JAK1 inhibitor Ruxolitinib before IFN-γ stimulation, it was found that the increased expression of M1-related marker genes caused by LAP3 deficiency was reversed after Ruxolitinib treatment, indicating that JAK1 played a key role in the polarization process of M1 macrophages. This shows that JAK1 is an important component of the JAK-STAT signaling pathway, which promotes the phosphorylation of STAT1 by mediating IFN-γ signaling, thereby enhancing the effector function of M1 macrophages. As a JAK1 inhibitor, Ruxolitinib can effectively block this signaling pathway, thereby inhibiting the polarization of M1 macrophages and the expression of related effector molecules. In summary, the experimental results further verified the key role of JAK1 in the polarization of M1 macrophages and showed the potential application value of Ruxolitinib in regulating this process. This provides new ideas and methods for future interventions targeting the JAK-STAT signaling pathway in the treatment of inflammatory and autoimmune diseases.

[0101] In summary, LAP3 can inhibit the activation of the JAK1-STAT1 signaling pathway and promote the activation of the STAT6 signaling pathway; the deletion of LAP3 does not change the expression of the IFN-γ membrane receptors IFN-γRα and IFN-γRβ; the promotion of M2 polarization of macrophages by LAP3 depends on STAT6 activity; the promotion of M1 polarization of macrophages by LAP3 deficiency depends on JAK1 activity.

[0102] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. Use of a substance for inhibiting or silencing LAP3 activity in the preparation of a medicament for treating tumors and / or delaying the invasion of Listeria, characterized in that, This is achieved by promoting the conversion of macrophages from the M2 phenotype to the M1 phenotype.

2. The use according to claim 1, characterized in that, The tumor is a solid tumor or a hematological tumor.

3. The use according to claim 2, characterized in that, The solid tumor is lung cancer, liver cancer, breast cancer, rectal cancer, pancreatic ductal adenocarcinoma, osteosarcoma, soft tissue sarcoma, glioblastoma, ovarian cancer or prostate cancer; The hematological tumor is lymphoma, multiple myeloma or leukemia.

4. The use according to claim 3, characterized in that, The tumor is lung cancer or liver cancer.

5. The use according to claim 4, characterized in that, The substance is one or more of nucleic acid, Bestatin or anti-LAP3 antibody.

6. The use according to claim 5, characterized in that, The nucleic acid is inhibitory nucleic acid.

7. The use according to claim 6, characterized in that, The inhibitory nucleic acid is miRNA, siRNA, shRNA, antisense RNA or lncRNA.

8. The use according to any one of claims 1-7, characterized in that, The drug further comprises a pharmaceutically acceptable excipient.

9. The use according to claim 8, characterized in that, The pharmaceutically acceptable excipient is any one or more of excipients, stabilizers, diluents, binders, preservatives, lubricants, antioxidants.

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