Application of MTHFD2 in reprogramming macrophage polarization by inhibiting PTEN

Abstract

The present invention discloses the application of MTHFD2 in reprogramming macrophage polarization by inhibiting PTEN. The present invention discovered for the first time that MTHFD2 inhibits the polarization of IFN-γ-activated macrophages in vitro and in vivo, but enhances the polarization of IL-4-activated macrophages, and determined that MTHFD2 is a regulator of macrophage polarization. Further experiments found that MTHFD2 leads to macrophage polarization reprogramming by inhibiting the PIP3 phosphatase activity of PTEN and enhancing the activation of downstream Akt. In addition, the present invention also found that myeloid deletion of MTHFD2 slowed tumor progression, promoted liver fibrosis, and reduced the recruitment of eosinophils. The present invention not only clarifies the previously unknown non-metabolic function of MTHFD2 in regulating PTEN activity, but also proposes a possible strategy for targeting MTHFD2 to regulate macrophage-mediated immune responses.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to application of MTHFD2 in reprogramming macrophage polarization by inhibiting PTEN. Background Art

[0002] In response to diverse microenvironmental stimuli, macrophages exert diverse functions in diverse physiological and pathological processes, including killing invading pathogens, tissue and organ remodeling and homeostasis, and tumor development. Macrophages can be polarized into two major activation phenotypes in vitro by T helper cytokines. M1 macrophages are typically activated by interferon (IFN)-γ and / or lipopolysaccharide (LPS) and possess cytotoxic, proinflammatory, and antitumor functions. M2 macrophages can be alternatively activated by interleukin-4 (IL-4) or interleukin-13 (IL-13) and possess helminth clearance, anti-inflammatory, and protumor functions. Macrophage polarization in vivo is even more complex. For example, tumor-associated macrophages (TAMs) are often plastic and heterogeneous within the M2 activation spectrum, promoting tumor progression by promoting angiogenesis and metastasis, immunosuppression, and even tumor chemoresistance. Therefore, it is of special significance to study the strategy of reprogramming tumor-associated macrophages (TAMs) from M2 macrophages to M1 macrophages.

[0003] Macrophage polarization is regulated by transcription factors, signal transduction, and metabolic reprogramming. STAT1 plays a key role in M1 macrophage M (IFN-γ) polarization, while key transcription factors involved in M2 macrophage M (IL-4) gene expression include STAT6, PPARγ, and PPARδ. Furthermore, M1 macrophages reprogram their metabolism to promote glycolysis and the pentose phosphate pathway, which in turn supports cytoplasmic reduced nicotinamide adenine diphosphate (NADPH) production, energy supply, and anabolism. Conversely, M2 macrophages reprogram their metabolism to increase mitochondrial oxidative metabolism. mTOR and Akt signaling have been reported to control macrophage metabolism and macrophage polarization. However, whether and how metabolic enzymes interact with canonical signal transduction pathways remains largely unexplored.

[0004] One-carbon (1C) metabolism involves the transfer of one-carbon units in the folate and methionine cycles, supporting the biosynthesis of glutathione (GSH), nucleotides, S-adenosylmethionine (SAM), and NADPH. In the cytosolic folate pathway, serine hydroxymethyltransferase 1 (SHMT1) transfers one carbon unit from the donor serine to tetrahydrofolate (THF), generating 5,10-methylenetetrahydrofolate. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) catalyzes the conversion of 5,10-methylenetetrahydrofolate back to tetrahydrofolate in two steps. Similar mitochondrial reactions are sequentially completed by serine hydroxymethyltransferase 2 (SHMT2), methylenetetrahydrofolate dehydrogenases MTHFD2 / 2L, and the methylenetetrahydrofolate dehydrogenase-like enzyme MTHFD1L. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) has higher catalytic activity than methylenetetrahydrofolate dehydrogenase 2-like (MTHFD2L) and can be induced in embryonic tissues and various tumors to meet higher biosynthetic demands. MTHFD2 is also reported to play a crucial role in the fate and function of immune cells, including T cells and B cells. However, whether and how MTHFD2 regulates macrophage polarization remains unclear. Summary of the Invention

[0005] One object of the present invention is to provide a novel use of a substance for inhibiting the activity of MTHFD2 protein or a substance for reducing the content of MTHFD2 protein.

[0006] The present invention provides use of a substance that inhibits the activity of MTHFD2 protein or a substance that reduces the content of MTHFD2 protein in preparing a product that promotes macrophage polarization to M1 type and / or inhibits macrophage polarization to M2 type.

[0007] In the above application, the substance that inhibits the activity of MTHFD2 protein or the substance that reduces the content of MTHFD2 protein inhibits the activation of Akt by enhancing the PIP3 phosphatase activity of PTEN, thereby promoting the polarization of macrophages to M1 type and / or inhibiting the polarization of macrophages to M2 type.

[0008] The present invention further provides the use of a substance that inhibits the activity of the MTHFD2 protein or a substance that reduces the content of the MTHFD2 protein in the preparation of a product for preventing and / or treating lung cancer.

[0009] The present invention also provides the use of a substance that inhibits the activity of the MTHFD2 protein or a substance that reduces the content of the MTHFD2 protein in the preparation of a product for inhibiting the growth and / or proliferation of lung cancer cells.

[0010] In any of the above applications, the substance that inhibits the activity of the MTHFD2 protein may be a protein, polypeptide or small molecule compound that inhibits the function of the MTHFD2 protein.

[0011] The substance that reduces the content of MTHFD2 protein may be a substance that inhibits the synthesis of MTHFD2 protein, promotes the degradation of MTHFD2 protein, or knocks down or knocks out the MTHFD2 gene.

[0012] Furthermore, the substance for knocking down the MTHFD2 gene may be siRNA or shRNA that inhibits the expression of the MTHFD2 gene.

[0013] The substance for knocking out the MTHFD2 gene may be an MTHFD2 gene editing system; the MTHFD2 gene editing system includes Cas9 nuclease and sgRNA targeting the MTHFD2 gene.

[0014] Furthermore, the nucleotide sequence of the siRNA that inhibits the expression of the MTHFD2 gene is specifically 5′-GCTCATGAAGAACACCATTA-3′.

[0015] Another object of the present invention is to provide a method for preparing M1 macrophages or a method for promoting the polarization of macrophages to M1.

[0016] The method for preparing M1 macrophages or promoting macrophage polarization to M1 provided by the present invention comprises the following steps: inhibiting the activity of MTHFD2 protein in macrophages or reducing the content of MTHFD2 protein in macrophages.

[0017] In the above method, the method for inhibiting the activity of the MTHFD2 protein in macrophages is to introduce the substance that inhibits the activity of the MTHFD2 protein into the macrophages.

[0018] The method for reducing the content of MTHFD2 protein in macrophages is to introduce the substance for reducing the content of MTHFD2 protein into the macrophages.

[0019] The use of the above method or the cells prepared according to the above method in screening drugs that regulate macrophage polarization also falls within the scope of protection of the present invention.

[0020] Another object of the present invention is to provide a product for regulating macrophage polarization or treating lung cancer or inhibiting the growth of lung cancer cells.

[0021] The active ingredient of the product for regulating macrophage polarization or treating lung cancer or inhibiting lung cancer cell growth provided by the present invention is the substance that inhibits the activity of MTHFD2 protein or the substance that reduces the content of MTHFD2 protein.

[0022] Another object of the present invention is to provide a method for constructing an animal model of liver fibrosis.

[0023] The method for constructing an animal model of liver fibrosis provided by the present invention comprises the following steps: knocking out the MTHFD2 gene in the myeloid system of an animal to obtain an MTHFD2 gene myeloid knockout animal (myeloid-specific MTHFD2 gene knockout animal), and inducing the MTHFD2 gene myeloid knockout animal (myeloid-specific MTHFD2 gene knockout animal) with CCl4 to obtain the animal model of liver fibrosis.

[0024] In the above method, compared with the liver fibrosis animal model obtained by directly inducing liver fibrosis in wild-type animals using CCl4, the liver fibrosis animal model constructed by the present invention exhibits more severe liver fibrosis, which is specifically manifested in increased levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum and / or expanded liver fibrosis area.

[0025] In the above method, the animal may specifically be a mouse.

[0026] The final object of the present invention is to provide the use of MTHFD2 protein as a target or the method for constructing the above-mentioned animal model of liver fibrosis, or the animal model of liver fibrosis constructed according to the above-mentioned method for constructing the animal model of liver fibrosis, in the preparation of products for improving liver fibrosis.

[0027] Any of the above macrophages can be mouse mononuclear macrophage leukemia cells RAW 264.7 or mouse bone marrow-derived macrophages.

[0028] Any of the above products may be a medicine.

[0029] Any of the above-mentioned M1 macrophages is an M1 macrophage activated by IFN-γ and / or LPS.

[0030] Any of the above-mentioned M2 macrophages is an IL-4 activated M2 macrophage.

[0031] The Gene ID of any of the above-mentioned MTHFD2 in NCBI is 17768.

[0032] The Gene ID of any of the above-mentioned PTENs in NCBI is 19211.

[0033] The Gene ID of any of the above-mentioned PI3Ks in NCBI is 18708.

[0034] The Gene ID of any of the above-mentioned Akt is 11651 in NCBI.

[0035] The present invention discovered for the first time that MTHFD2 inhibits the polarization of IFN-γ-activated macrophages (M(IFN-γ)) in vitro and in vivo, but enhances the polarization of IL-4-activated macrophages (M(IL-4)), confirming that MTHFD2 is a regulator of macrophage polarization. Further experiments revealed that MTHFD2 leads to macrophage polarization reprogramming by inhibiting the PIP3 phosphatase activity of PTEN and enhancing downstream Akt activation. In addition, the present invention also found that myeloid deletion of MTHFD2 slows tumor progression, promotes liver fibrosis, and reduces the recruitment of eosinophils. The present invention not only clarifies the previously unknown non-metabolic function of MTHFD2 in regulating PTEN activity, but also proposes a possible strategy for targeting MTHFD2 to regulate macrophage-mediated immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 MTHFD2 inhibits M (IFN-γ) and promotes M (IL-4) polarization. Data are presented as mean ± SEM. n = 3 per group (A–C, E, G, and H). NS, not significant (p ≥ 0.05). * p<0.05, ** p<0.01, *** p<0.001, Student's t-test.

[0037] Figure 2 MTHFD2 regulates tumor-associated macrophage polarization in vivo. Data are presented as mean ± SEM. n = 8 per group (A); n = 5 per group (B); n = 6 per group (CE). * p<0.05, ** p<0.01, *** p<0.001, Student's t-test.

[0038] Figure 3 Impaired M2 polarization in MTHFD2 myeloid-deficient mice. Data are presented as mean ± SEM. n = 3 per group (A, E, F, and G); n = 4-6 per group (C, D). * p<0.05, ** p<0.01, *** p<0.001, Student's t-test.

[0039] Figure 4 MTHFD2 enhances Akt activation by reducing the PIP3 phosphatase activity of PTEN. Data are presented as mean ± SEM. n = 3 / group (C–E); n = 6 per group (F). NS, not significant (p ≥ 0.05). * p<0.05, ** p<0.01, *** p<0.001, Student's t-test. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0041] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0042] The mouse experimental materials and preparation methods involved in the following examples are as follows:

[0043] All mice were of C57BL / 6 background and maintained under specific pathogen-free conditions at the Tianjin Medical University Animal Laboratory under a 12-h light / 12-h dark cycle at a controlled temperature (22°C ± 2°C). Unless otherwise specified, mice were fed a standard diet (#1010049, Synergy Biological) and free access to clean water. All animal experiments were approved by the Ethics Committee of Tianjin Medical University.

[0044] Mthfd2 fl / fl Mice (C57BL / 6 mice with loxP sequences inserted flanking the Mthfd2 gene): bred by Cyygen Biosciences (Guangzhou, China) using CRISPR-Cas9 technology.

[0045] Lyz2-Cre mice are described in the literature “Shen, L., et al., Serine metabolism antagonizesantiviral innate immunity by preventing ATP6V0d2-mediated YAP lysosomal degradation. Cell Metab., 2021.33(5): p.971-987e6”.

[0046] Mthfd2 fl / fl Lyz2-Cre - Mice: Mthfd2 fl / fl The mice were hybridized with Lyz2-Cre mice, and mice and their offspring containing loxP sequences but not Lyz2-Cre genes were selected from the hybrid offspring through genotyping.

[0047] Mthfd2 fl / fl Lyz2-Cre+ Mice: Mthfd2 fl / fl Mice were hybridized with Lyz2-Cre mice, and myeloid-specific Mthfd2 gene knockout mice and their offspring containing loxP sequences and Lyz2-Cre genes were selected from the hybrid offspring through genotyping.

[0048] All experimental groups used age- and sex-matched mice aged 6-12 weeks. The genotypes of the mice were determined by PCR analysis of mouse tail DNA using the following primers:

[0049] Mthfd2lox-F:5'-TTTTCAGCCACCGAGACCATCCT-3';

[0050] Mthfd2lox-R:5'-GACAACGGCTTCATTTCTGCAG-3';

[0051] Lyz2-cre-F:5'-CCGAAATGCAATTACG-3';

[0052] Lyz2-cre-R:5'-TCTGGCTGCAGAATTTCTG-3'.

[0053] The experimental methods involved in the following examples are as follows:

[0054] CCl4-induced liver fibrosis model: 1.0 mL / kg of CCl4 (diluted 1:3 with corn oil) was intraperitoneally administered to 8-12 weeks old mice. and Mice with liver fibrosis (n=5 per group) were injected 3 times a week for a total of 12 times.

[0055] Chitin model: Chitin (Sigma) was sonicated on ice and diluted in 30 mL PBS. and Mice (n=5 per group) were intraperitoneally injected with chitin (3 μg).

[0056] Tumor xenograft model: 6 weeks old and Mice (n=5 per group) were injected with 5×10 6 Murine Lewis lung carcinoma cells LLC cells (murine Lewis lung carcinoma cells ls).

[0057] Bone marrow isolation and cell culture: from 8-12 weeks old and Bone marrow was isolated from mice and lysed using erythrocyte lysis buffer (#R1010, Solarbio). The cells were cultured for 7 days in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin (P / S), and 30% L929-conditioned medium to differentiate into bone marrow-derived macrophages (BMDMs). The medium was then replaced to remove non-adherent cells. In polarization experiments, BMDMs were stimulated with 100 ng / mL IFN-γ (50709-MNAH, Sino Biological) or 20 ng / mL IL-4 (404-ML, R&D Systems) for the indicated times.

[0058] Isolation and Cell Culture of Peritoneal Macrophages: Primary peritoneal macrophages (PMs) were collected from C57BL / 6 mice by injecting them with 1 ml of sulfur gel (3:100; #225650, BD). Peritoneal fluid was carefully aspirated from the peritoneal cavity without puncturing any organs. Cells were centrifuged for 10 minutes and resuspended in RPMI-1640 medium supplemented with 10% FBS. PMs were cultured at 37°C for 1-2 hours to allow them to adhere to 6-well plates. PMs were stimulated using the same protocol as for BMDMs.

[0059] Cell lines: Human embryonic kidney (HEK293T) cells and mouse Lewis lung carcinoma (LLC) cells were cultured in DMEM (Hyclone, #SH30243.01) supplemented with 10% fetal bovine serum and 1% polysaccharide (PS). Mouse monocytic macrophage leukemia (RAW 264.7) cells were cultured in RPMI 1640 medium at 37°C in a humidified atmosphere of 5% CO₂. All cell lines were confirmed to be mycoplasma-free.

[0060] Transfection and Stimulation: siRNAs were transfected into BMDMs and RAW 264.7 cells using RNAi Max (Thermo Fisher Scientific) according to the manufacturer's instructions. BMDMs were incubated with 20 μM SC79 (HY-18749, MCE) for 2 h to activate Akt 1. Bone marrow cells were cultured in macrophage differentiation medium for 3 days and then infected every 2 days with lentivirus carrying either empty vector or encoding MTHFD2. Four days after infection, BMDMs were selected with 1 μg / mL puromycin and used for further experiments.

[0061] FACS immunofluorescence analysis: and Immunofluorescence staining of mouse splenocytes was performed. Spleens were minced into single cells, and splenocytes were collected and lysed in red blood cell lysis buffer (#R1010, Solarbio). Each sample was incubated with F4 / 80, CD11b, CD45, or CD170 antibodies on ice for 30 minutes in the dark. Cells were collected on a FACSFortessa flow cytometer (BD), and data were analyzed using FlowJo software (Tree Star).

[0062] Immunofluorescence and Immunohistochemistry Analysis: For immunofluorescence, liver tissue or xenograft tumors were fixed with 4% PFA at 4°C and washed with PBS. Tissues were equilibrated in 30% sucrose for 1 h, then in 30% sucrose:OCT (1:1) overnight at 4°C and arranged in cryostat molds containing 100% OCT. The entire cryostat was frozen in a dry ice bath, and cryosections were prepared. Frozen liver sections were incubated with F4 / 80 or ARG1 antibodies and probed with fluorescent secondary antibodies. Frozen xenograft tumor sections from C57BL / 6J mice were incubated with IL-6, IL-1β, or iNOS antibodies and probed with fluorescent secondary antibodies. For immunohistochemistry, xenograft tumors from C57BL / 6J mice were fixed with 4% PFA, dehydrated with graded ethanol, and then embedded in paraffin. Tissues in 6-μm sections were dewaxed and rehydrated. Antigen retrieval was performed by boiling tissue sections in 10 mM sodium citrate buffer (pH 6.0) for 15 minutes in a microwave oven. Nonspecific binding sites were blocked by incubating sections with 10% donkey serum for 1 hour at room temperature (RT). Xenograft tumor sections from C57BL / 6J mice were incubated with rat monoclonal antibodies against CD11b, CD4, CD8α, FOXP3, F4 / 80, iNOS, or ARG1. After incubation with the primary antibody, sections were washed with TBST and incubated with biotinylated goat anti-mouse / rabbit / rat IgG antibodies for 2 hours at room temperature. Following washing, sections were incubated with HRP-streptavidin for 1 hour at room temperature. Following staining with 3,3′-diaminobenzidine (DAB), sections were counterstained with hematoxylin, mounted, and imaged using a Nikon upright microscope. RAW264.7 cells were seeded onto confocal microscopy plates to detect expression of HA-PTEN and Flag-tagged MTHFD2. Cells were fixed with 4% PFA for 10 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 10 minutes at 4°C. After incubation with 3% BSA in PBST (PBS + 0.1% Tween 20) for 30 minutes to block nonspecific binding, cells were incubated with anti-Flag (rabbit, Thermo Fisher Scientific) and anti-HA (mouse, BioLegend) antibodies overnight at 4°C, followed by incubation with anti-rabbit Alexa Fluor 488 (Invitrogen) and anti-mouse DyLight 633 (Invitrogen) secondary antibodies for 2 hours at room temperature. HEK-293A cells expressing Flag-tagged MTHFD2 or Flag-tagged N-terminal deletion mutants of MTHFD2 were treated with 100 nM mitochondrial probe (Yeasen) and incubated for 30 minutes. The cells were then fixed, permeabilized, stained with anti-Flag antibody, and incubated with Alexa Fluor 488 antibody (Invitrogen).After the immunofluorescence procedure, cells were stained with 0.5 μg / mL DAPI for 5 min, washed with PBS, and photographed using a Zeiss LSM800 confocal microscope.

[0063] Immunoprecipitation and immunoblotting: Proteins were extracted from cultured cells or tissues using cell lysis buffer, followed by immunoprecipitation and immunoblotting using appropriate antibodies. (Cell lysis was performed using RIPA lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitors. Equal amounts of protein were separated by SDS-PAGE and analyzed by immunoblotting using specific antibodies to determine target protein levels.) Mitochondrial proteins were isolated using a cell mitochondrial isolation kit (Beyotime). Proteins from cell lysates were subjected to SDS-PAGE, then transferred to PVDF membranes (Bio-Rad) and probed with the indicated antibodies, including ARG1, iNOS, MTHFD2, α-Tubulin, p-Akt (S473), p-Akt (T308), Akt, PTEN, p85, p-STA T6 (Y641), STAT6, p-STAT1 (Y701), STAT1, p-FOXO1 (S256), FOXO1, Flag, p-p70 S6K (T389), S6K, p-mTOR (S2448), mTOR, HA-HRP, Hsp60, YY1, and β-actin-HRP. Immunoblots were visualized using a chemiluminescence imaging system (Tanon).

[0064] RNA extraction and quantitative real-time PCR: Total RNA from BMDM and RAW264.7 cells was extracted using TRIzol RNA isolation reagent (Invitrogen) and reverse transcribed into cDNA using TransScript II First-Strand cDNA Synthesis SuperMix (TransGen). Quantitative real-time PCR was performed using SYBR Master Mix (Yeasen) and the designed primers. –ΔΔCt Methods: Fold changes in gene expression were calculated, and β-actin expression was used as an internal control. The qPCR primer sequences were as follows:

[0065] MTHFD2-F:TGGCTGCGACTTCTCTAATG.

[0066] MTHFD2-R:CCTTCCAGAAATGACAACAGC.

[0067] β-actin-F:CGTACCACTGGCATCGTGAT.

[0068] β-actin-R:AGGTAGTCAGTCAGGTCCCG.

[0069] Mus-Mthfd2-F:AGTGCGAAATGAAGCCGTTG.

[0070] Mus-Mthfd2-R:GACTGGCGGGATTGTCACC.

[0071] Mus-iNOS-F:GAAACGCTTCACTTCCAATG.

[0072] Mus-iNOS-R:AATCCACAACTCGCTCCAA.

[0073] Mus-IL-6-F:TTGCCTTCTTGGGACTGAT.

[0074] Mus-IL-6-R:TTGCCATTGCACAACTCTT.

[0075] Mus-IL-1β-F:GAAATGCCACCTTTTGACAGTG.

[0076] Mus-IL-1β-R:TGGATGCTCTCATCAGGACAG.

[0077] Mus-Arg1-F:CCACAGTCTGGCAGTTGGAAG.

[0078] Mus-Arg1-R:GGTTGTCAGGGGAGTGTTGATG.

[0079] Mus-Retnla-F:CCAATCCAGCTAACTATCCCTCC.

[0080] Mus-Retnla-R:ACCCAGTAGCAGTCATCCCA.

[0081] PTEN phosphatase assay: Cells with the indicated stimuli were collected for PTEN phosphatase assay. and BMDM from mice were stimulated with IL-4 for 0, 10, 30, and 60 minutes. BMDM transfected with HA-PTEN and Flag-tagged MTHFD2 or a Flag-tagged MTHFD2 N-terminal deletion mutant were harvested. HA-tagged PTEN or HA-PTENΔ118–141 and MTHFD2 or MTHFD2Δ215–225 were expressed in RAW 264.7 cells. Cells were repeatedly frozen and thawed with 50 mM HEPES and 150 mM NaCl. PTEN or HA-PTEN was immunoprecipitated from the lysate using a PTEN antibody or anti-HA agarose beads (Sigma, A2095) with rotation overnight at 4°C. The beads were washed three times in cell lysis buffer and incubated for 30 minutes at 30°C with 25 μM PIP3 (Cayman, 10007764), 10 mM MgCl2, and 50 mM HEPES (pH 7.0). For in vitro PTEN protein phosphatase assays, purified PTEN was preincubated with MTHFD2 peptide (aa 215–225) for 20 min and then reacted with PIP3 in the above buffer. Phosphate release was determined by measuring absorbance at 620 nm using a malachite green phosphate assay kit (Sigma, MAK307).

[0082] PI3K activity: BMDM were stimulated with IL-4 for 0, 10, 30, and 60 min. Cells were lysed and immunoprecipitated with p85 antibodies before incubation with Protein A+G agarose (Beyotime). PI3K activity was measured using an Echelon ELISA kit.

[0083] Biotin-streptavidin pull-down assay: Biotinylated PTEN peptide (aa 118–141) or a biotinylated control peptide was incubated with streptavidin beads at 4°C for 2 hours with rotation, followed by incubation with purified MTHFD2 or 293T lysate at 4°C overnight. The beads were then washed, and immunoblotting was performed to detect MTHFD2 expression. Similarly, biotinylated MTHFD2 peptide (aa 215–225) or a biotinylated control peptide was incubated with purified PTEN protein or lysate from 293T cells overexpressing HA-PTEN, and immunoblotting was performed to detect PTEN expression.

[0084] Quantification and Statistical Analysis: Data are presented as mean ± SEM. Statistical significance was set at p < 0.05. Significance between two groups was determined by Student's t-test (unpaired two-tailed, unequal variances). Statistical analyses were performed in GraphPad Prism 7. Unless otherwise noted, all experiments were repeated at least three times. Sample numbers are indicated in the figure legends.

[0085] The meanings of the professional terms involved in the following embodiments are as follows:

[0086] M(IFN-γ): M1 macrophages.

[0087] M(IL-4): M2 macrophages.

[0088] The Gene ID of MTHFD2 in the following examples is 17768 in NCBI.

[0089] The Gene ID of PTEN in the following examples is 19211 in NCBI.

[0090] The Gene ID of PI3K in the following examples is 18708 in NCBI.

[0091] The Gene ID of Akt in the following examples is 11651 in NCBI.

[0092] Example 1: MTHFD2 inhibits M (IFN-γ) and promotes M (IL-4) polarization

[0093] 1. Effect of siRNA knockdown of MTHFD2 in macrophages on macrophage polarization

[0094] siControl: siControl (siControl: 5′-UUCUCCGAACGUGUCACGU-3′) was introduced into RAW 264.7 cells using RNAiMax (Thermo Fisher Scientific), and control cells were obtained after identification.

[0095] siMTHFD2: siMTHFD2 (siMTHFD2: 5′-GCTCATGAAGAACACCATTA-3′) was introduced into RAW 264.7 cells using RNAi Max (Thermo Fisher Scientific), and MTHFD2 knockdown cells were obtained after identification.

[0096] qRT-PCR was used to detect the MTHFD2 knockdown efficiency in control cells and MTHFD2 knockdown cells.

[0097] Control cells and MTHFD2 knockdown cells were stimulated with IFN-γ (100 ng / ml) for 12 h, and then the expression of M1 markers was detected by qRT-PCR. Cells without IFN-γ were used as a control.

[0098] Control cells and MTHFD2 knockdown cells were stimulated with IL-4 (20 ng / ml) for 24 h, and then the expression of M2 markers was detected by qRT-PCR. No IL-4 was added as a control.

[0099] The results showed that inhibition of MTHFD2 by specific interfering RNA (siRNA) significantly promoted the expression of M1 marker genes iNOS and interleukin-6 (IL-6) in RAW 264.7 cells, and inhibited the expression of M2 marker genes Arg1 and Retnla in RAW 264.7 cells. Figure 1 A and 1B).

[0100] Effects of CRISPR-Cas9 knockout of MTHFD2 in macrophages on macrophage polarization

[0101] Test cells:

[0102] MTHFD2-WT: derived from Mthfd2 fl / fl Lyz2-Cre - Mouse bone marrow-derived macrophages.

[0103] MTHFD2-KO: derived from Mthfd2 fl / fl Lyz2-Cre + Mouse bone marrow-derived macrophages.

[0104] MTHFD2-WT or MTHFD2-KO BMDMs were stimulated with IFN-γ (100 ng / ml) for 12 h or for the indicated times, and the expression of M1 markers was detected by qPCR or western blotting. BMDMs without IFN-γ were used as controls.

[0105] MTHFD2-WT or MTHFD2-KO BMDMs were stimulated with IL-4 (20 ng / ml) for 24 h or for the indicated times, and then the expression of M2 markers was detected by qPCR or western blotting. BMDMs without IL-4 were used as controls.

[0106] The results showed that compared with MTHFD2-WT macrophages, the expression of M1 marker genes was upregulated and the expression of M2 marker genes was downregulated in MTHFD2-KO macrophages ( Figure 1 C–1F).

[0107] Effects of overexpression of MTHFD2 in MTHFD2-knockout macrophages on macrophage polarization

[0108] Test cells:

[0109] MTHFD2-WT: derived from Mthfd2 fl / fl Lyz2-Cre- Mouse bone marrow-derived macrophages.

[0110] MTHFD2-KO: derived from Mthfd2 fl / fl Lyz2-Cre + Mouse bone marrow-derived macrophages.

[0111] The MTHFD2 expression plasmid was introduced into MTHFD2-WT or MTHFD2-KO BMDMs, and then the expression of M1 markers was detected by qPCR or WB after stimulation with IFN-γ (100 ng / ml) for 12 h, or the expression of M2 markers was detected by qPCR or WB after stimulation with IL-4 (20 ng / ml) for 24 h.

[0112] The MTHFD2 expression plasmid was obtained by cloning MTHFD2 cDNA into the plenti-GFP-Flag vector (OBiO Technology) digested with EcoRI and BamHI, while keeping other sequences of the plenti-GFP-Flag vector unchanged.

[0113] The results showed that overexpression of MTHFD2 in RAW 264.7 cells significantly reduced the expression of M1 marker genes and enhanced the expression of M2 marker genes ( Figure 1 G and 1H). In addition, MTHFD2 overexpression rescued the macrophage polarization phenotype of MTHFD2-KO primary macrophages ( Figure 1 G and 1H). These results indicate that MTHFD2 regulates macrophage polarization.

[0114] Example 2: MTHFD2 regulates tumor-associated macrophage polarization in vivo

[0115] exist and Mice were subcutaneously injected with 2×10 6 LLC cells (mouse lung cancer cells) were used to construct LLC xenograft tumor models. The tumor volume (volume = width) was measured and calculated every 2 days starting from the 4th day after inoculation. 2 × length / 2). Observe tumor conditions and measure tumor weight 21 days after inoculation. and Tumor sections of mice were stained with immunofluorescence and semi-quantified histologically.

[0116] The results showed that: Compared with mice, The tumor volume and weight of mice were significantly reduced, and tumor growth was reduced ( Figure 2A and 2B), indicating that MTHFD2 knockout can inhibit lung cancer cell growth. Next, immunohistochemical staining of tumor sections was used to detect immune cell infiltration in the tumor. CD11b in tumor sections + 、CD4 + and CD8a + The average cell number ratio The number of Treg cells increased significantly by nearly 2-fold, while the number of Treg cells decreased to about 50% of the control ( Figure 2 C). Further investigation of macrophage infiltration and polarization in tumor sections revealed that the number of F4 / 80-positive macrophages increased by nearly 2-fold ( Figure 2 D). The number of cells expressing iNOS was significantly upregulated, while the number of cells expressing ARG1 was downregulated ( Figure 2 D). In addition, immunofluorescence staining showed that the proportion of macrophages expressing IL-1β, iNOS or IL-6 was significantly increased ( Figure 2 E), showing that MTHFD2 knockout promoted M(IFN-γ) polarization and inhibited M(IL-4) polarization of macrophages in vivo.

[0117] Example 3: Impaired M2 polarization in MTHFD2 myeloid-deficient mice

[0118] 1. and Mice were injected with CCl4 for one month to construct and A mouse model of liver fibrosis was constructed. After model establishment, liver tissue was collected for biochemical and histochemical analysis. Frozen liver sections were prepared and stained with Oil Red O to observe tissue fatty degeneration. Serum AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels were measured according to the manufacturer's instructions (Nanjing Jiancheng). Liver RNA was extracted, and the expression of relevant genes was detected by real-time fluorescence quantitative PCR (qPCR).

[0119] The results showed that myeloid MTHFD2-deficient mice showed more severe liver fibrosis and increased serum AST and ALT levels ( Figure 3 A) Expansion of fibrosis area ( Figure 3 B and 3C).

[0120] two, and Chitin was injected into the peritoneal cavity of the mouse model of liver fibrosis, and CD45 in the peritoneal cavity of the mouse was detected by flow cytometry 2 days later. + The percentage of eosinophils or the total number of eosinophils in the cells were measured, and the expression of Arg1 mRNA in mouse peritoneal macrophages was detected by qPCR.

[0121] The results showed that myeloid MTHFD2 deficiency reduced eosinophil recruitment and decreased ARG1 expression in macrophages. Figure 3 E-3G), indicating that MTHFD2 deficiency impairs the function of M(IL-4) in vivo, which further suggests that MTHFD2 plays an important role in promoting the function of M(IL-4) macrophages in vivo.

[0122] Example 4: MTHFD2 enhances Akt activation by reducing the PIP3 phosphatase activity of PTEN, thereby leading to macrophage polarization reprogramming

[0123] I. MTHFD2-WT or MTHFD2-KO BMDMs were stimulated with IL-4 (20 ng / ml) for the indicated times and then subjected to immunoblotting analysis using the indicated antibodies.

[0124] The results showed that Akt in MTHFD2-KO BMDM S473 and Akt T308 The background and IL4-induced phosphorylation of Akt and the phosphorylation of downstream Akt targets FoxO1 and S6K1 were significantly attenuated ( Figure 4 A).

[0125] Second, MTHFD2-WT or MTHFD2-KO BMDMs were pre-stimulated with or without 100 μM SC79 for 2 h, then stimulated with IL-4 (20 ng / ml) for 15 min, and then analyzed by immunoblotting using the antibodies shown in the figure.

[0126] The results showed that SC79 pre-stimulation led to the expression of Akt in MTHFD2-KO BMDM S473 and downstream Akt targets showed increased background and IL4-induced phosphorylation levels ( Figure 4 B).

[0127] Third, MTHFD2-WT or MTHFD2-KO BMDMs were pre-stimulated with or without 100 μM SC79 for 2 h, and then stimulated with IFN-γ (100 ng / ml) for 12 h or IL-4 (20 ng / ml) for 24 h. The expression of M1 markers or M2 markers was detected by qPCR.

[0128] The results showed that Akt signaling is crucial for the abnormal polarization caused by MTHFD2 deficiency. Figure 4 C and 4D).

[0129] Fourth, PI3K activity was measured in anti-p85 immunoprecipitates from unstimulated or IL-4-stimulated BMDMs, and PIP3 phosphatase activity of PTEN in IL-4-stimulated BMDMs was measured by ELISA. The amount of PIP3 was normalized to the amount of p85 in the immunoprecipitate, which was determined by Western blot densitometry. The amount of released phosphate was normalized to the amount of immunoprecipitated PTEN quantified by Western blot densitometry.

[0130] The results showed that the expression and activity of PI3K in IL-4-stimulated MTHFD2-KO and WT cells were comparable. The expression level of PTEN was also comparable in both cell types. However, under unstimulated and IL-4-stimulated conditions, the PIP3 phosphatase activity of PTEN in MTHFD2-deficient cells was much higher than that in WT cells ( Figure 4 E and 4F).

[0131] V. MTHFD2-WT or MTHFD2-KO BMDMs were transfected with siControl (siControl: 5′-UUCUCCGAACGUGUCACGU-3′) or siPTEN (siPTEN: 5′-CGACUUAGACUUGACCUAUAU-3′) using RNAi Max (Thermo Fisher Scientific) and then stimulated with IL-4 for the indicated times and subjected to immunoblotting analysis with the indicated antibodies.

[0132] The results showed that siRNA knockdown of PTEN significantly enhanced Akt phosphorylation in unstimulated and IL-4-stimulated MTHFD2-deficient cells ( Figure 4 G).

[0133] In summary, MTHFD2 enhances Akt activation by reducing the PIP3 phosphatase activity of PTEN, leading to macrophage polarization reprogramming.

[0134] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for preparing M1 macrophages, characterized in that: The method comprises the following steps: inhibiting the activity of MTHFD2 protein in macrophages or reducing the content of MTHFD2 protein in macrophages.

2. A method for promoting macrophage polarization to M1 type, characterized in that: The method comprises the following steps: inhibiting the activity of MTHFD2 protein in macrophages or reducing the content of MTHFD2 protein in macrophages.

Images