Method for inducing polarization of macrophages by AKK and / or outer membrane vesicles thereof and application of AKK and / or outer membrane vesicles thereof in preparation of medicine for promoting phagocytosis of infected red blood cells by macrophages

Abstract

The invention discloses a method for inducing polarization of macrophages by AKK and / or outer membrane vesicles thereof and application of AKK and / or outer membrane vesicles thereof in preparation of drugs for promoting phagocytosis and infection of erythrocytes by macrophages, and belongs to the technical field of biological medicines. According to the invention, after macrophages are induced by AKK bacteria or OMVs thereof, an M1 polarization phenomenon occurs. Macrophages polarized by M1 generate proinflammatory factors, and meanwhile, erythrocytes infected by plasmodium are subjected to more phagocytosis. Compared with macrophages not polarized by M1, the macrophages polarized by M1 induced by AKK bacteria or AKK-OMVs can swallow more erythrocytes infected by plasmodium under the condition of not using the existing anti-malarial drugs, so that the resistance of plasmodium to the anti-malarial drugs due to long-term use of the anti-malarial drugs is effectively avoided; and a new scheme is provided for clinical malaria treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to a method for inducing macrophage polarization using Akkermansia muciniphila (AKK) and / or its outer membrane vesicles and its application in preparing a drug for promoting macrophage phagocytosis of infected red blood cells. Background Art

[0002] Malaria is one of the three major global public health problems that seriously threaten human health. Antimalarial drugs, including quinine, chloroquine, mefloquine, sulfadoxine / pyrimethamine, artemisinin, and piperaquine, are the first-line treatments for malaria. However, long-term use of antimalarial drugs can lead to genetic mutations in Plasmodium parasites, conferring resistance to these drugs and significantly reducing their effectiveness. As drug resistance intensifies, researchers are exploring new antimalarial drugs. For example, several new artemisinin derivatives and non-artemisinin drugs are undergoing laboratory, preclinical, and clinical trials. The development of these compounds requires long drug development cycles, high investment, and a very low success rate. Based on the experience with Plasmodium parasite resistance to both former first-line antimalarial drugs (quinine, chloroquine, mefloquine, sulfadoxine / pyrimethamine) and the current first-line antimalarial drug (artemisinin), even if new drugs are successfully used in antimalarial treatment, drug resistance will likely continue for the foreseeable future due to the genetic mutations these compounds can induce in Plasmodium parasites under drug pressure. Therefore, bypassing the development of new compounds as antimalarial drugs is a new way to circumvent the development of drug resistance in Plasmodium.

[0003] Prior art CN119367402A discloses the use of Akkermansia muciniphila in improving and treating cerebral malaria caused by Plasmodium berghei. In a mouse cerebral malaria model, the effects of Akkermansia muciniphila alone or in combination with adjuvant drugs (dihydroartemisinin, rapamycin, and atorvastatin) were evaluated to improve and treat cerebral malaria in mice. The results suggest that whether used alone or in combination with drugs, the bacteria can effectively improve mouse behavior, immune pathological damage to various organs, reduce parasitemia, and prolong mouse survival. Compared with using drugs alone, it can effectively treat cerebral malaria in mice.

[0004] Prior art CN111840250B discloses a novel agent that can be used to treat malignant cerebral malaria. The agent is composed of a hollow protein shell formed by self-assembly of 24 protein subunits and an iron-based nanozyme with catalase activity. It can specifically target brain microvascular endothelial cells and scavenge ROS. First, because the agent's protein shell has iron ion channels, nano-iron cores with uniform particle size can be synthesized in its cavity. The nano-iron core has catalase catalytic activity and can catalyze the decomposition of hydrogen peroxide. Secondly, the ferritin shell can target brain endothelial cells, while promoting the proliferation of macrophages in the liver and polarization to the M1 subtype, enhancing their phagocytic function of infected red blood cells. Therefore, this agent can be used to treat cerebral malaria.

[0005] Prior art CN111918967B discloses a composition and method comprising extracellular vesicles containing nucleic acids that target genes and induce macrophage polarization of tumor-associated macrophages, as well as the use of extracellular vesicles in preparing a composition for regulating gene expression in macrophages.

[0006] Prior art CN117264871A discloses the use of Artemisia annua extracellular vesicles in drugs for regulating the immune microenvironment. Artemisia annua extracellular vesicles are used as immunomodulators and co-cultured with macrophages to polarize macrophages in vivo and in vitro from an M2-like phenotype to an M1 phenotype.

[0007] Macrophages are one of the primary effector cells of the innate immune system and play a crucial role in innate immunity. Macrophages can eliminate pathogens by phagocytosis. Activated macrophages enhance their ability to phagocytose pathogens. Therefore, enhancing macrophage phagocytosis of red blood cells infected with Plasmodium parasites is an effective approach to address antimalarial drug resistance. Summary of the Invention

[0008] The present invention aims to provide the use of Akkermansia muciniphila (AKK) and / or its outer membrane vesicles in the preparation of a medicament for treating diseases caused by Plasmodium, thereby overcoming the problems of the prior art. The present invention discloses a composition comprising Akkermansia muciniphila and its outer membrane vesicles, which induces polarization of macrophages M1, and the polarized macrophages enhance phagocytosis of red blood cells infected with Plasmodium.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] One of the technical solutions of the present invention is a method for inducing macrophage polarization using Akkermansia muciniphila (AKK) and / or its outer membrane vesicles, which comprises the step of inducing macrophage polarization using Akkermansia muciniphila (AKK) and / or its outer membrane vesicles.

[0011] In certain embodiments, the polarization is a reversal from an unpolarized M0 state to an M1 polarized state or from an M2 polarized state to an M1 polarized state.

[0012] In certain embodiments, the Akkermansia muciniphila is Akkermansia muciniphila JCM 33894T strain, purchased from Japan Collection of Microorganisms (JCM).

[0013] In certain embodiments, the M1 polarized macrophages produce pro-inflammatory factors and phagocytose more Plasmodium-infected erythrocytes.

[0014] The second technical solution of the present invention is an M1 polarized macrophage, wherein the M1 polarized macrophage is prepared by the above method.

[0015] The third technical solution of the present invention is the use of Akkermansia muciniphila (AKK) and / or its outer membrane vesicles in the preparation of a drug for promoting macrophages to phagocytose infected red blood cells.

[0016] A fourth technical solution of the present invention is the use of Akkermansia muciniphila (AKK) and / or its outer membrane vesicles in the preparation of an antimalarial drug-resistant composition.

[0017] A fifth technical solution of the present invention is a use of Akkermansia muciniphila (AKK) and / or its outer membrane vesicles in preparing a composition for regulating gene expression in macrophages.

[0018] A sixth technical solution of the present invention is a use of the above-mentioned M1 polarized macrophages in the preparation of an antimalarial drug-resistant composition.

[0019] Based on the above technical solution, the present invention has the following technical effects:

[0020] The present invention discovered that after macrophages were induced by AKK bacteria or their OMVs, M1 polarization occurred. The polarized M1 macrophages produced pro-inflammatory factors and simultaneously phagocytosed more infected red blood cells. The present invention can phagocytose red blood cells infected with Plasmodium without the use of drugs, relying solely on polarized macrophages. This method effectively avoids the long-term use of antimalarial drugs, which can cause Plasmodium to develop resistance to antimalarial drugs. The infected red blood cells do not need to be cleared with drugs, and the pathogen is cleared by phagocytosis, effectively avoiding the occurrence of drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Figure 3 shows the morphology of AKK bacteria and AKK-OMVs. (A) Gram-stained image of AKK bacteria under a light microscope, scale bar: 10 μm; (B) morphology of AKK bacteria under a transmission electron microscope (TEM), scale bar: 500 nm; (C) morphology of AKK-OMVs under a transmission electron microscope, scale bar: 200 nm.

[0023] Figure 2 Observation for protozoaemia.

[0024] Figure 3 RT-qPCR verification of the effects of AKK and AKK-OMVs on macrophage polarization. A represents the relative expression of iNOS mRNA, B represents the relative expression of CD86 mRNA, C represents the relative expression of TNF-α mRNA, D represents the relative expression of Arg-1 mRNA, E represents the relative expression of CD206 mRNA, and F represents the relative expression of TGF-β mRNA.

[0025] Figure 4 RT-qPCR validation of the reverse polarization of M2-polarized macrophages by AKK and AKK-OMVs. A represents the relative expression of iNOS mRNA, B represents the relative expression of CD86 mRNA, C represents the relative expression of TNF-α mRNA, D represents the relative expression of Arg-1 mRNA, E represents the relative expression of CD206 mRNA, and F represents the relative expression of TGF-β mRNA. G represents the mass spectrometry analysis of AKK-OMV components.

[0026] Figure 5 To observe the effect of AKK and AKK-OMVs on macrophage polarization by flow cytometry.

[0027] Figure 6 The flow cytometry was used to observe the effect of AKK and AKK-OMVs on the reverse polarization of M2 macrophages.

[0028] Figure 7Giemsa-stained smears were used to observe the phagocytosis of infected red blood cells (iRBCs) by macrophages after 1 hour of co-incubation. A is the BLK group (blank group), B is the IFN-γ + LPS group (classical M1 polarization group), C is the IL-4 group (classical M2 polarization group), D is the AKK group, and E is the AKK-OMVs group. F is the phagocytosis rate of infected RBCs by macrophages in each group.

[0029] Figure 8 Observation of AKK bacteria and AKK-OMV-induced macrophage M1 polarization and phagocytosis of infected erythrocytes using different microscopes. (AC) Observation of AKK bacteria-induced macrophage M1 polarization and phagocytosis of infected erythrocytes using optical, scanning, and transmission electron microscopy, respectively. (DF) Observation of AKK-OMV-induced macrophage M1 polarization and phagocytosis of infected erythrocytes using optical, scanning, and transmission electron microscopy, respectively.

[0030] Figure 9 Results of the macrophage clearance and replenishment experiment. A shows the weight of mice in the BLK, NEG, M0, M1, and M2 groups from D0 to D7; B shows the RMCBS score of mice in the BLK, NEG, M0, M1, and M2 groups from D2 to D7; and C shows the parasitemia of mice in the BLK, NEG, M0, M1, and M2 groups from D0 to D6. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0037] The embodiments of the present invention provide the use of Akkermansia muciniphila and / or its outer membrane vesicles in the preparation of a drug for treating diseases caused by Plasmodium.

[0038] In some specific embodiments, the Akkermansia muciniphila JCM 33894T strain was purchased from the Japan Collection of Microorganisms (JCM).

[0039] In some specific embodiments, the Plasmodium comprises Plasmodium berghei strain ANKA.

[0040] In some specific embodiments, Akkermansia muciniphila promotes the polarization of macrophages M1 to phagocytose Plasmodium-infected erythrocytes.

[0041] An embodiment of the present invention further provides a drug for treating diseases caused by Plasmodium, comprising Akkermansia muciniphila and / or its outer membrane vesicles.

[0042] The embodiments of the present invention also provide use of Akkermansia muciniphila and / or its outer membrane vesicles in preparing a drug for promoting macrophage M1 polarization.

[0043] The embodiments of the present invention also provide use of Akkermansia muciniphila and / or its outer membrane vesicles in preparing a drug for reversing macrophage M2 polarization.

[0044] In the present invention, RBCs refers to normal red blood cells; iRBCs refers to red blood cells infected with Plasmodium, also referred to as infected red blood cells or infected red blood cells.

[0045] Example 1

[0046] 1 Materials and Methods

[0047] Experimental Animals, Plasmodium Strains, and Insect Strains: Female C57BL / 6 mice (weighing 18–22 g, 8–10 weeks old) were purchased from HNSJA Co., Ltd., Changsha, China. Mice were housed under specific pathogen-free conditions and fed a UV-irradiated diet and purified water to maintain appropriate living and feeding conditions (25 ± 3°C). All mice were acclimated to the environment 1 week prior to the experiment.

[0048] The Plasmodium berghei strain ANKA (PbA) was kindly donated by Professor Xu Wenyue of the Army Medical University and was subcultured and frozen in liquid nitrogen for long-term storage in our laboratory.

[0049] Akkermansia muciniphila JCM 33894T strain was purchased from Japan Collection of Microorganisms (JCM).

[0050] The experimental instruments are shown in Table 1.

[0051] Table 1 Experimental instruments

[0052] name model Origin and company OLYMMPUS microscope BX53 Olympus Corporation, Japan centrifuge 5424R Eppendorf, Germany Constant temperature metal bath MK-20 China, Aosheng Micropipette Eppendorf, Germany Mouse tail vein injection instrument KW-XXY China, Calvin refrigerator -20℃，-80℃ Haier, China Fluorescence quantitative PCR instrument CFXOPUS 96 Bio-Rad, USA flow cytometer CytoFLEX Beckman Coulter, USA Ultracentrifuge Optima XE100 Beckman Coulter, USA

[0053] Experimental reagents and consumables are shown in Table 2.

[0054] Table 2 Experimental reagents and kits

[0055] name Manufacturer Giemsa stain China, Rain and Dew syringe Weigao, China Cryogenic tubes Thermo Fisher Scientific, USA slides China, Shitai Methanol China, Zhongtian dimethyl sulfoxide VWR, USA Slide storage box China, Blue Sky PBS buffer powder UK, Servicebio Pipette tips Thermo Fisher Scientific, USA Fluorescently labeled antibodies BioLegend, USA Primers Azenta, USA Immersion oil China, Source Leaf Sodium heparin micro-blood collection tube China, Kangshifei

[0056] 2AKK bacterial culture and extracellular vesicle preparation

[0057] (1) AKK bacterial culture

[0058] Culture medium (3.85 g brain heart infusion, 1.6 g soy peptone, 1.13 g anhydrous glucose, 0.55 g N-acetylglucosamine, 0.4 g L-threonine, 0.05 g L-cysteine, 100 mL) was anaerobically cultured at 37°C for 3 days until the bacterial solution became slightly turbid. After counting, the bacterial precipitate was collected by centrifugation, washed, and resuspended in PBS for later use.

[0059] (2) Preparation of AKK extracellular vesicles

[0060] Centrifuge 400 mL of the expanded AKK culture at 11,000 g for 20 minutes, and collect the supernatant. Aliquot the supernatant into 50 mL centrifuge tubes, centrifuge at 11,000 g for 10 minutes, collect the supernatant, and centrifuge again at 11,000 g for 10 minutes. Pass the supernatant through a 0.45 μm filter and a 0.22 μm filter using a vacuum filtration device. Centrifuge at 200,000 g for 130 minutes in an ultracentrifuge. Discard the supernatant to precipitate visible AKK-OMVs. Resuspend the AKK-OMVs in PBS, determine their concentration using the BCA assay, and store frozen at -80°C.

[0061] 3 Establishment of mouse malaria model

[0062] (1) Recovery and passage: Place the PbA malarial parasite strain frozen in liquid nitrogen in a 37°C water bath. After it is thawed, draw blood with a 1ml syringe and immediately inoculate it intraperitoneally into C57BL / 6 mice at a dose of 0.2ml / mouse. This is the recovery of the parasite strain. Prepare a blood smear from the tip of the tail of the blood-producing mouse. Count the percentage of infected red blood cells in 1000 red blood cells in different fields of view, which is the parasitemia density. When the parasitemia level reaches 15%-30%, remove the eyeball and draw blood in a heparin anticoagulant tube. 6 The iRBCs-infected erythrocytes were passaged into offspring C57BL / 6 mice at a dose of 0.2 mL / mouse.

[0063] (2) Establishment of PbA infection model in mice: When the parasitemia level of donor mice reached 15%-30%, the eyeballs were removed and blood was collected in heparin anticoagulant tubes. 6 The iRBCs were inoculated into experimental mice at a dose of 0.2 ml / mouse to establish a PbA infection mouse model.

[0064] (3) Observation of parasitemia: Blood was collected from the tail vein and thin blood smears were prepared and stained with Giemsa. Prositemia was assessed on the Giemsa-stained thin blood smears under a light microscope with a 10x eyepiece and a 100x objective. Prositemia was examined and quantified by counting the number of iRBCs in at least 1,000 red blood cells.

[0065] 4 Observation of iRBCs phagocytosis by macrophages in vitro

[0066] (1) Inducing macrophage polarization trend

[0067] Use 2 μL of 10 9 CFU / mLAKK or 80 μL 250 μg / mLAKK extracellular vesicles with 2×10 5 BV2 macrophages were co-cultured in 6-well plates at 37°C and 5% CO2 to induce their polarization.

[0068] Subsequently, 10 μL of RBCs and iRBCs were added to the culture system respectively.

[0069] Subsequently, BV2 cells were collected after thorough lysis with Trizol and subjected to RT-qPCR using primer pairs for six indicators: TNF-α, TGF-β, CD86, CD206, iNOS, and Arg. The cell pellets from the co-culture were collected, incubated with fluorescently labeled antibodies against F4 / 80, CD86, and CD206, and analyzed by flow cytometry. Comparison with a control group containing only BV2 cells revealed the polarization trend of BV2 cells during the immune response in the co-culture.

[0070] (2) Statistical analysis of the phagocytic rate of iRBCs by macrophages

[0071] The phagocytic rate of iRBCs by BV2 cells was detected and calculated by flow cytometry. Far Red-incubated BV2 cells and treated iRBCs were mixed in a 1.5 mL centrifuge tube with serum-free medium according to the proportion, co-cultured at 37°C, 5% CO2 for 2 hours, centrifuged and the supernatant discarded, and washed twice with PBS to remove residual fluorescent dye. Each group of samples was processed according to the flow cytometry method and loaded onto the machine. The APC-positive cell group was circled in the cell population, which was the BV2 cell group. Then, the FITC-positive cell group was circled in the BV2 cell group, which was the BV2 cell group that phagocytosed iRBCs. The ratio of the two cell groups calculated by the software was the ratio of BV2 cells that underwent phagocytosis.

[0072] 5. Observation of iRBCs phagocytosis by macrophages in vivo

[0073] 6-8 week old C57BL / 6 mice were randomly divided into 4 groups, 5 mice in each group. After acclimation, each mouse was intraperitoneally injected with 200 μL of passaged mouse erythrocytes with a parasite density of 15%, and the day was designated as D0.

[0074] On the same day, each mouse in the BLK group was intraperitoneally injected with 200 μL of liposome PBS solution without clodronate;

[0075] Each mouse in the NEG, M0, and M1 groups was intraperitoneally injected with 200 μL of clodronate liposomes to eliminate macrophages in the body.

[0076] Starting from D0, the weight of mice needs to be recorded every day.

[0077] On D3, each mouse in the BLK group was intraperitoneally injected with 200 μL of liposome PBS solution without clodronate;

[0078] The NEG group continued to receive 200 μL of clodronate liposomes (the same as D0) and the M0 group received 10 6Each mouse in the M1 group was intraperitoneally injected with 10 6 BV2 cells in the M1 polarized state after treatment with 200 μL AKK-OMVs.

[0079] Starting from D3, blood smears were prepared every day to count the parasitemia of each group, and the body weight of each group was measured regularly and the RMCBS score was performed until the mice began to die one after another on D6.

[0080] 5 Experimental results

[0081] 5.1 Successful culture of AKK bacteria and extraction of AKK-OMVs

[0082] After three passages and expanded culture, AKK-OMVs with a concentration of 8 mg / mL were obtained by filtration and ultra-high-speed centrifugation, as determined by the BCA method. Figure 2 At the same time, AKK bacteria in the logarithmic growth phase were frozen and used for subsequent experiments.

[0083] 5.2 Successful establishment of a mouse model of Plasmodium infection

[0084] Resuscitate the ANKA strain of Plasmodium berghei (PbA) frozen in liquid nitrogen and infect 6-8 week old female C57BL / 6 mice. Count the parasitemia on blood smears. Passage the mice when the parasitemia is >15%. Use the third passage of mice as experimental subjects. If the parasitemia is >15% and accompanied by typical neurological symptoms, the model is successfully established. Figure 2 .

[0085] 5.3 RT-qPCR verification of the polarization and repolarization effects of AKK and its extracellular vesicles on macrophages

[0086] 5.3.1 RT-qPCR Verification of Macrophage Polarization

[0087] In the co-culture experiment, the statistical difference analysis of the relative expression levels between the groups showed that the experimental group with the addition of AKK bacteria and AKK-OMVs showed a trend of macrophage M1 polarization, and AKK-OMVs had a stronger ability to induce macrophage M1 polarization than AKK bacteria. Figure 3 As shown, target genes indicating M1 are iNOS, CD86, and TNF-α, while target genes indicating M2 are Arg, CD206, and TGF-β. AKK bacteria and AKK-OMVs can inhibit the expression of M2-related genes, and the expression levels of M2-related genes in the AKK-OMV group are lower.

[0088] 5.3.2 RT-qPCR Verification of Macrophage Reverse Polarization

[0089] After confirming that AKK bacteria or AKK-OMVs can polarize macrophages to M1, a reversal experiment was designed to verify whether macrophages can be transformed from M2 to M1 under the action of AKK bacteria or AKK-OMVs. After giving macrophages an M2 polarized culture environment, AKK and AKK-OMVs were added. According to the analysis of the relative expression levels of different target genes, AKK and AKK-OMVs still have different degrees of M1 polarization conversion effects on macrophages that have completed M2 polarization, and AKK-OMVs have a better reversal effect. Among them, AKK-OMVs mass spectrometry analysis shows that they contain multiple active ingredients such as Amuc_1100, which may affect the function and activity of macrophages, such as Figure 4 shown.

[0090] 5.4 Flow cytometry verification of the polarization and repolarization effects of AKK and AKK-OMVs on macrophages

[0091] 5.4.1 Flow cytometry verification of the macrophage polarization effect of AKK and AKK-OMVs

[0092] After co-culture as above, BV2 cells were collected, CD86 and CD206 were used as fluorescent dyes, and after incubation, they were placed on a flow cytometer. The results showed that the BV2 cells in the AKK group and the AKK-OMVs group were all M1 polarized, which was consistent with the RT-qPCR results. Figure 5 shown.

[0093] 5.4.2 Flow cytometry verification of the reverse polarization effect of AKK and AKK-OMVs on M2 macrophages

[0094] After co-culture as above, BV2 cells were collected, CD86 and CD206 were used as fluorescent dyes, and after incubation, they were placed on a flow cytometer. The results showed that AKK and AKK-OMVs in each group had a tendency to reverse the M2 polarized BV2 cells to M1 polarization, and AKK-OMVs were more obvious, which was consistent with the RT-qPCR results. Figure 6 shown.

[0095] 5.5 Observation of macrophage phagocytosis in vitro and calculation of phagocytic rate

[0096] 5.5.1 Morphological observation under optical microscope

[0097] After co-culture, BV2 cells were collected by centrifugation as above. Six smears containing thick and thin blood films were prepared for each group. After drying, they were fixed with formaldehyde for 20 seconds. Freshly prepared Giemsa stain (Giemsa:PBS=1:10) was evenly dripped onto the smears. The staining was timed for 10 minutes. The stain was slowly rinsed with a thin stream of water, avoiding direct contact with the smears. After continuous rinsing for 1 minute, the stain was allowed to dry. The phagocytosis of macrophages in each smear was observed. Figure 750 fields of view were randomly photographed for each smear for phagocytosis statistics. The phagocytosis rates of the AKK group and the AKK-OMVs group were significantly higher than those of the CTL group. Compared with the AKK group, there was no significant difference in the phagocytosis of iRBCs in the AKK-OMVs group, suggesting that AKK-OMVs can be used instead of AKK bacteria to induce macrophage M1 polarization and promote the latter to phagocytose iRBCs. Prepare unstained phagocytic smears of co-culture, such as Figure 7 shown.

[0098] 5.5.2 Morphological observation of phagocytosis under scanning electron microscope Figure 8 -A, B, C. These figures show optical microscopy, scanning electron microscopy, and transmission microscopy images of BV2 cells phagocytosing iRBCs after AKK treatment.

[0099] 5.5.3 Morphological observation under transmission electron microscope is shown in the figure. Figure 8 -D, E, F. These figures show optical microscopy, scanning electron microscopy, and transmission microscopy images of BV2 cells phagocytosing iRBCs after the action of AKK-OMVs.

[0100] 5.6 In vivo observation of phagocytosis of infected erythrocytes by polarized macrophages

[0101] As the number of days of infection increased, all experimental groups except the BLK group showed varying degrees of weight loss on D1 after the injection of macrophage scavengers, but the weight of most experimental groups recovered on D2. After D3, the weight of all C57BL / 6 mice showed a downward trend, among which the decline in the M1 group was relatively stable, while the decline in the BLK group became larger in the later period. The RMCBS score showed a downward trend from D3, and the score dropped significantly after D4. The score of the M1 group did not show a significant decline until D5. Prositemia was recorded from D3, and the parasitemia of the group injected with macrophage scavengers was slightly higher than that of the BLK group. The upward trend of the M1 group was relatively slow, the BLK group died on D6, and the NEG group, M0 group and M1 group died on D7, respectively. Figure 9 .

[0102] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for inducing macrophage polarization using AKK and / or its outer membrane vesicles, characterized in that: The method comprises the steps of inducing macrophage polarization using AKK and / or its outer membrane vesicles; The AKK is Akkermansia muciniphila; The polarization is reversed from the M2 polarization state to the M1 polarization state.

2. The method according to claim 1, characterized in that The Akkermansia muciniphila is Akkermansia muciniphila JCM 33894T strain, purchased from Japan Collection of Microorganisms (JCM).

3. The method according to claim 1, characterized in that The M1 polarized macrophages produced pro-inflammatory factors and phagocytosed more red blood cells infected with Plasmodium.

4. Use of AKK and / or its outer membrane vesicles in the preparation of a drug for promoting macrophage phagocytosis of infected red blood cells, wherein the AKK is Akkermansia muciniphila.

5. Use of AKK and / or its outer membrane vesicles in preparing an antimalarial drug-resistant composition, wherein the AKK is Akkermansia muciniphila.

6. Use of AKK and / or its outer membrane vesicles in preparing a composition for regulating gene expression in macrophages, wherein the AKK is Akkermansia muciniphila.

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