Development and application of mouse model for simulating pneumonia mononuclear-derived macrophage recruitment

Abstract

The invention provides development and application of a mouse model for simulating pneumonia mononuclear-derived macrophage recruitment, and belongs to the technical field of biological medicine. A mouse expressing human ACE2 in the lung is constructed through an adeno-associated virus (AAV) carrier, then 2-3 mg / kg of spike protein is atomized to the lung through aerosolization, the modeling time is 3 days, and the process of novel coronavirus infection diseases can be better simulated. By focusing the index that mononuclear macrophages are infiltrated into the lung in quantity, the phenotype that mononuclear macrophages recruitment from the lung in pneumonia is successfully simulated on a mouse. The method is low in operation difficulty and simple in steps. The method has a good application prospect in the development of drugs for regulating / targeting mononuclear macrophage recruitment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to development and application of a mouse model simulating recruitment of pneumonia mononuclear-derived macrophages. BACKGROUND

[0002] During the epidemiological evolution of the novel coronavirus, mutations occur continuously, and multiple variants are derived, including Alpha, Beta, Delta, and Omicron. Adaptive mutations exist in the genomes of these variants, which help the virus to evade the host immune response and lead to an increase in reinfection rate.

[0003] An ideal mouse model can be used for the evaluation of the therapeutic effect of novel coronavirus treatment drugs, the testing of vaccine effectiveness, and the pathogenic mechanism of the novel coronavirus, and can help virus prevention and control. The receptor for the invasion of the novel coronavirus into human cells is human angiotensin-converting enzyme 2 (hACE2), and the mouse homologous receptor mouse ACE2 (mACE2) cannot mediate the invasion of the virus due to differences in key amino acid sites, so the construction of a mouse model needs to overcome the difficulty that mice are not naturally susceptible to the novel coronavirus.

[0004] In terms of operation, most of the existing technologies use tracheal instillation or intratracheal atomization. The former is a invasive operation, which is difficult to operate and has the risk of infection. The latter is an invasive operation, which is difficult to operate and can easily cause suffocation and death of the mouse during the operation.

[0005] In terms of time, the modeling time of the existing technology is mostly 24 hours, which aims to simulate the acute lung injury caused by the novel coronavirus. However, the flow results show that compared with other respiratory viruses such as influenza virus, the lung injury caused by the novel coronavirus infection is slower in onset, but progresses rapidly, lasts longer, and leads to higher hospitalization mortality. The modeling time of about 3 days is closer to the real disease progression of the novel coronavirus infection.

[0006] The existing technology also discloses the use of a mixture of spike proteins derived from the novel coronavirus and lipopolysaccharide (LPS), spike proteins and poly(i:c) as a reagent for the construction of a pneumonia mouse model, which aims to simulate lung inflammatory injury. LPS is a bacterial component, and poly(i:c) is structurally similar to double-stranded RNA (dsRNA), but neither of them is a component inherent to the novel coronavirus, which may lead to a deviation in the judgment of researchers in the evaluation of the therapeutic effect of novel coronavirus treatment drugs, the testing of vaccine effectiveness, and the pathogenic mechanism of the novel coronavirus.

[0007] Winkler ES et al. found that after hACE2 knock-in (KI) mice, these mice express hACE2 under the endogenous promoter instead of mouse ACE2 (mACE2). Intranasal inoculation of hACE2 KI mice with the novel coronavirus WA1 / 2020 led to extensive replication of the virus in the upper and lower respiratory tract. However, the hACE2 KI mice infected with the novel coronavirus did not lose weight and had limited pathology, and this model could only serve as a model of mild infection of the strain. (Winkler ES, Chen RE, Alam F, et al. Novel coronavirus Causes Lung Infection without Severe Disease in Human ACE2 Knock-In Mice. J Virol. 2022; 96(1): e0151121.).

[0008] Another group used adeno-associated virus (AAV) to express ACE2 in C57BL / 6J mice. Although infectious particles can be recovered from mice infected with the novel coronavirus, they all do not show any clinical symptoms of novel coronavirus infection, and no mice have significant body weight changes or deaths (Israelow B, Song E, Mao T, Lu P, Meir A, Liu F, Alfajaro MM, Wei J, Dong H, Homer RJ, Ring A, Wilen CB, Iwasaki A. Mouse model of novel coronavirus reveals inflammatory role of type I interferon signaling. J Exp Med. 2020 Dec 7; 217(12): e20201241. doi: 10.1084 / jem.20201241. Erratum in: J Exp Med. 2025 Apr 7; 222(4): e2020124102192025c. ), so it is also not a suitable animal model. SUMMARY

[0009] In order to solve the above problems, the present application provides a mouse model simulating the recruitment of pulmonary monocyte-derived macrophages and its development and application.

[0010] In one aspect, the present application provides a method for constructing a mouse model simulating the recruitment of pulmonary monocyte-derived macrophages, comprising the following steps: (1) administering AAV-hACE2 to the mouse by pulmonary aerosolization; (2) administering the spike protein to the mouse by pulmonary aerosolization; The AAV-hACE2 is an AAV vector expressing hACE2; and the spike protein is a full-length or fragment of spike protein derived from coronavirus.

[0011] Specifically, in step (1), the AAV includes but is not limited to at least one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R or AAVrh1.

[0012] Preferably, the AAV is AAV9.

[0013] Specifically, in step (1), the AAV-hACE2 is administered at an amount of 3.16×10 10 -3.16×10 12 GC per mouse.

[0014] Further specifically, the AAV-hACE2 is administered at an amount of 3.0×10 11 -3.5×10 11 GC per mouse.

[0015] Specifically, in step (2), the spike protein is a full-length spike protein derived from coronavirus.

[0016] Specifically, in step (2), the spike protein is administered at an amount of 2-6 mg / kg.

[0017] Further specifically, the spike protein is administered at an amount of 2-3 mg / kg.

[0018] Specifically, in step (2), the spike protein is aerosolized at a condition of a median particle size <10 μm.

[0019] Further specifically, the spike protein is aerosolized at a condition of a median particle size <5 μm.

[0020] In another aspect, the present application provides a mouse model prepared by the above construction method, and application of the mouse model in screening, evaluation or quality control of a drug for regulating / recruiting monocyte-derived macrophages.

[0021] In still another aspect, the present application provides a method for screening, evaluating or quality controlling a drug for regulating / recruiting monocyte-derived macrophages, comprising the following steps: S1, constructing a mouse model according to the above construction method; S2, administering the drug to be tested to the mouse model. S3, detecting the indicators of the model is used for screening, evaluating or quality control of drugs.

[0022] Preferably, in S2, the drug is a pneumonia drug; further preferably, a drug for pneumonia caused by the novel coronavirus.

[0023] Specifically, in S2, the administration method comprises at least one of oral administration, injection, implantation, external use, spraying, and inhalation.

[0024] Further specifically, the administration method is nebulization inhalation or intravenous injection.

[0025] Preferably, the administration method is nebulization inhalation.

[0026] Specifically, in S3, the indicators include at least one of the proportion of CD11b+F4 / 80+ monocyte-derived macrophages, the infiltration of CD11b+F4 / 80+ monocyte-derived macrophages in the lung, and the concentration of inflammatory factor protein.

[0027] Further specifically, the inflammatory factor comprises at least one of IL-6, IL-1β, TNF-α, and IFN-γ.

[0028] Compared with the prior art, the present application has the following advantages: The modeling method provided by the present application has low operation difficulty and simple steps. The constructed mouse model has obvious clinical symptoms, and the mouse has obvious weight loss but does not die. The white blood cells and monocytes increase significantly, the inflammatory factors such as IL-6 and IL-1β also increase significantly, a large number of inflammatory infiltrates can be seen in the lung tissue, and the alveolar structure collapses and other pathological damages. The mouse model constructed by the present application provides an in vivo evaluation method for the development of drugs for regulating / recruiting monocyte-derived macrophages. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a schematic diagram of the mouse model construction process in Example 1.

[0030] Figure 2 It is the Western blot detection of the expression level of hACE2 protein in the lung of the mouse after being given AAV-hACE2 in Example 1, and GAPDH is used as an internal reference.

[0031] Figure 3 It is the particle size distribution of the liquid droplets ultrasonically atomized detected by the particle size analyzer in Example 1, and the median particle size is 3.471 μm.

[0032] Figure 4 It is a curve graph of the body weight change of the mice in each group from the 0th day to the 7th day of modeling in Example 1.

[0033] Figure 5 For the flow cytometry detection of the proportion of CD11b+F4 / 80+ monocyte-derived macrophages in the bronchoalveolar lavage fluid of mice in each group in Example 1.

[0034] Figure 6 For the quantitative analysis of the change in the proportion of CD11b+F4 / 80+ monocyte-derived macrophages in the bronchoalveolar lavage fluid of mice in each group in Example 1, **** represents p<0.0001.

[0035] Figure 7 For the cell counting analysis to detect the number of white blood cells in the bronchoalveolar lavage fluid of mice in each group in Example 1, * represents p<0.05; ** represents p<0.01; and *** represents p<0.001.

[0036] Figure 8 For the cell counting analysis to detect the number of monocytes in the bronchoalveolar lavage fluid of mice in each group in Example 1, ** represents p<0.01.

[0037] Figure 9 For the ELISA detection of the IL-6 protein concentration level in the bronchoalveolar lavage fluid of mice in each group in Example 1, *** represents p<0.001; and **** represents p<0.0001.

[0038] Figure 10 For the ELISA detection of the IL-1β protein concentration level in the bronchoalveolar lavage fluid of mice in each group in Example 1, * represents p<0.05; ** represents p<0.01; and *** represents p<0.001.

[0039] Figure 11 For the hematoxylin-eosin (H&E) staining of lung tissue sections of mice in each group in Example 1, the scale bar is 100 μm.

[0040] Figure 12 For the morphology of MSCs-Exo before and after nebulization taken by transmission electron microscopy in Example 2, the scale bar is 200 nm.

[0041] Figure 13 For the detection of the influence of different administration methods on the distribution of MSCs-Exo in different organs by small animal live imaging instrument in Example 2.

[0042] Figure 14 For the flow cytometry detection of the proportion of CD11b+F4 / 80+ monocyte-derived macrophages in the bronchoalveolar lavage fluid of mice in each group in Example 2.

[0043] Figure 15For the quantitative analysis of the change in the proportion of CD11b+F4 / 80+ monocyte-derived macrophages in the bronchoalveolar lavage fluid of each group of mice in Example 2, ** represents p<0.01.

[0044] Figure 16 For the immunohistochemical staining of lung tissue sections of each group of mice in Example 2, the indicators are CD11b and F4 / 80.

[0045] Figure 17 For the ELISA detection of IL-6 protein concentration levels in the bronchoalveolar lavage fluid of each group of mice in Example 2.

[0046] Figure 18 For the ELISA detection of IL-1β protein concentration levels in the bronchoalveolar lavage fluid of each group of mice in Example 2.

[0047] Figure 19 For the hematoxylin-eosin (H&E) staining of lung tissue sections of each group of mice in Example 2, the scale is 100 m. DETAILED DESCRIPTION

[0048] The present application will be further described in conjunction with specific examples, which are not intended to limit the present application, but only serve to illustrate the present application. Unless otherwise specified, the experimental methods used in the following examples are generally in accordance with conventional conditions. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial sources.

[0049] For example, the mice used in the examples are C57BL / 6J, 6 weeks old, male, and weigh 18-23 g. However, those skilled in the art can adjust the above specific limitations by conventional techniques based on the schemes described in the present application, and achieve the same effect as the schemes described in the examples.

[0050] Example 1 Construction of a mouse model simulating monocyte-derived macrophage recruitment in pneumonia In this example, first, a mouse expressing human ACE2 in the lung was constructed by an adeno-associated virus (AAV) vector. Then, a certain amount of spike protein was aerosolized and administered to the lungs of the experimental group mice by aerosolization. At different time points (3 days, 7 days), the lung monocyte-derived macrophage infiltration and lung inflammation damage of the mice were observed, and the modeling scheme was finally determined. Figure 1 ).

[0051] To construct the experimental group of mice expressing human ACE2 in the lung, first, 3.16×10 11AAV-hACE2 (purchased from Guangzhou Pishen Biotechnology), the control group was given the same titer of AAV-EGFP, after 3 weeks, the lung tissue of the mice was taken, RIPA lysis buffer was added and ground into tissue homogenate, then the total protein of the lung tissue was extracted, and Western blot was used to detect the expression of hACE2 protein. The results showed that compared with the control group, the characteristic band of hACE2 could be observed in the AAV-hACE2 group, indicating that the lung expression of human ACE2 in mice was successfully constructed Figure 2 ). The mice were randomly divided into experimental and control groups.

[0052] Then, an ultrasonic atomizer was used to atomize 2.5 mg / kg of spike protein (Yiqiaoshenzhou, item number 40589-V08B1) into the lungs of the experimental mice. The liquid droplets atomized by the ultrasonic atomizer were fine and uniform, and the median particle size was controlled below 5 μm, ensuring that the modeling drug could enter and deposit in the small bronchi Figure 3 ).

[0053] After the administration of spike protein, the weight change curves of mice on different days were recorded. The results showed that compared with the control group, the body weight of the experimental mice decreased significantly in the first 3 days and reached the lowest on the 3rd day, and then gradually recovered, which suggested that the pathological damage caused by spike protein in mice might reach the peak on the 3rd day, and then the mice gradually recovered Figure 4 ).

[0054] Therefore, we collected the bronchoalveolar lavage fluid (BALF) and lung tissue of mice on the 0th, 3rd and 7th days, and performed pathological detection. Flow cytometry was used to detect the cells in BALF, and the results showed that the proportion of monocyte-derived macrophages in BALF increased significantly on the 3rd day and decreased significantly on the 7th day compared with the 3rd day, which indicated that after the administration of spike protein, it would cause a large number of monocyte-derived macrophages to recruit to the lungs, and reach the peak on the 3rd day, and then gradually decrease Figures 5-6 ). Similarly, the analysis of the number of cells in BALF found that the number of white blood cells Figure 7 ) and monocytes Figure 8 ) in BALF increased significantly on the 3rd day and decreased significantly on the 7th day compared with the 3rd day. ELISA kit was used to detect the inflammatory factors such as IL-6 (Elabscience, E-EL-M0044) and IL-1β (Elabscience, E-EL-M0037) in BALF, and the results showed that the protein concentrations of IL-6 Figure 9 ) and IL-1β Figure 10 ) in BALF also increased significantly on the 3rd day and decreased significantly on the 7th day compared with the 3rd day.

[0055] Next, we performed hematoxylin-eosin (H&E) staining on mouse lung tissue sections to observe the pathological condition of the lung tissue. Hematoxylin can stain the nucleus blue-purple, and eosin can stain the cytoplasm red or pink. The results showed that compared with the control group, a large number of inflammatory infiltrates and alveolar structure collapse were observed in the lung tissue of the mice in the 3-day group, while the pathological damage such as inflammatory infiltrates and alveolar structure collapse in the lung tissue of the mice in the 7-day group was alleviated. The above results show that by giving 2.5 mg / kg of spike protein through ultrasonic nebulizer pulmonary nebulization, the median particle size of the nebulized droplets is controlled below 5 μm, and the modeling time is 3 days, which can simulate the large recruitment of monocyte-derived macrophages in the lung and inflammatory lung injury Figure 11 ).

[0056] Example 2 Effect verification of mouse model in lung inflammation drug screening Using the mouse model constructed in Example 1, the therapeutic effect of MSCs-Exo on regulating the recruitment of monocyte-derived macrophages to alleviate pneumonia was detected. MSCs-Exo was isolated and purified from the culture supernatant of primary mesenchymal stem cells, and existing technologies have confirmed its therapeutic effect on pneumonia (Lotfy A, AboQuella NM, Wang H. Mesenchymal stromal / stem cell (MSC)-derived exosomes in clinical trials. Stem Cell Res Ther. 2023;14(1):66. Published 2023 Apr 7. doi:10.1186 / s13287-023-03287-7). Since the novel coronavirus mainly infects the respiratory tract and lungs, nebulization is one of the most suitable administration methods for treating pneumonia.

[0057] First, the ability of the ultrasonic nebulizer to nebulize MSCs-Exo was detected: the nebulized liquid was collected, and the basic morphology of the exosomes before and after nebulization was observed by transmission electron microscopy. The results showed that tea cup-shaped exosomes could be observed in the nebulized liquid, indicating that ultrasonic nebulization did not destroy the basic structure of the exosomes Figure 12 ).

[0058] Next, Cy5.5 dye was used to incubate exosomes overnight to stain the exosomes, and then these stained exosomes were administered to ordinary mice through different administration routes to observe the effect of different administration routes on the in vivo distribution of exosomes. After 24 h of administration to mice, small animal live imaging detection found that exosomes administered by intravenous injection were mostly enriched in the liver, while exosomes administered by pulmonary nebulization were mostly still enriched in the lungs, with only a few circulating to the liver Figure 13 ), which indicates that pulmonary nebulization can effectively deliver drugs to the lungs.

[0059] After 6h of modeling, the model mice were treated with 1x10 11 MSCs-Exo by atomization, and the bronchoalveolar lavage fluid (BALF) and lung tissue of the mice in each group were collected at the third day of treatment for detection. Flow cytometry was used to detect the cells in BALF, and the results showed that the proportion of CD11b+F4 / 80+ monocyte-derived macrophages in BALF was significantly reduced after atomization of MSCs-Exo Figures 14-15 . Immunohistochemical staining of lung tissue sections showed that atomization of MSCs-Exo could significantly reduce the infiltration of CD11b+F4 / 80+ monocyte-derived macrophages in the lungs Figure 16 . Similarly, ELISA was used to detect inflammatory factors such as IL-6 and IL-1β in BALF, and the results showed that the protein concentration of inflammatory factors in BALF was significantly reduced in the treatment group of atomization of MSCs-Exo Figures 17-18 . Finally, H&E staining of lung tissue sections showed that atomization of MSCs-Exo could significantly alleviate inflammatory infiltration and protect alveolar structure compared with the model group Figure 19 . The above results show that the mouse model developed by the application to simulate the recruitment of monocyte-derived macrophages in lung inflammation provides an in vivo evaluation system for drugs targeting the regulation / recruitment of monocyte-derived macrophages.

[0060] It should be noted that the above content is only used to illustrate the technical solutions of the application, and is not a limitation on the protection scope of the application. Simple modifications or equivalent replacements of the technical solutions of the application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the application.

Claims

1. A method for constructing a mouse model simulating the recruitment of mononuclear-derived macrophages in pneumonia, characterized in that, Includes the following steps: (1) AAV-hACE2 was administered to mice via pulmonary nebulization; (2) Administering spike protein to mice via pulmonary nebulization; The AAV-hACE2 is an AAV vector expressing hACE2; the spike protein is the full length or a fragment of the spike protein derived from coronavirus.

2. The construction method according to claim 1, characterized in that, In step (1), the AAV is selected from at least one of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, or AAVrh1.

3. The construction method according to claim 1, characterized in that, In step (1), the dosage of AAV-hACE2 is 3.16 × 10⁻⁶. 10 -3.16×10 12 GC.

4. The construction method according to claim 1, characterized in that, In step (2), the amount of spike protein administered is 2-6 mg / kg.

5. The construction method according to claim 1, characterized in that, In step (2), the atomization conditions for the spike protein are: median particle size < 10 μm.

6. A mouse model of simulated pneumonia mononuclear-derived macrophage recruitment constructed according to the construction method of any one of claims 1-5.

7. The use of the mouse model prepared by the construction method according to any one of claims 1-5 in the screening, evaluation or quality control of drugs that regulate / target the recruitment of mononuclear-derived macrophages.

8. A method for screening, evaluating, or quality controlling drugs that regulate / target the recruitment of monocyte-derived macrophages, characterized in that, Includes the following steps: S1. Construct a mouse model using the construction method according to any one of claims 1-5; S2. Administer the test drug to the mouse model; S3. Detect various indicators in mouse models for drug screening, evaluation, or quality control.

9. The method according to claim 8, characterized in that, In S3, the indicators include at least one of the following: the proportion of CD11b+F4 / 80+ mononuclear macrophages, the infiltration of CD11b+F4 / 80+ mononuclear macrophages in the lungs, and the concentration of inflammatory cytokine proteins.

10. The evaluation method according to claim 9, characterized in that, The inflammatory factors include at least one of IL-6, IL-1β, TNF-α, and IFN-γ.

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