Brucella-influenza A virus co-infection mouse model and its construction method and application

Abstract

The application provides a Brucella virus-influenza virus A co-infection mouse model and a construction method and application thereof, and belongs to the technical field of mouse model construction.The construction method of the Brucella virus-influenza virus A co-infection mouse model provided by the application is that after the mouse is anesthetized, the mouse is infected with the influenza virus A by using a lung delivery method, and 10-14 hours later, the mouse is infected with the Brucella by using the lung delivery method.The Brucella virus-influenza virus A co-infection mouse model is constructed, which is used for exploring the synergistic pathogenic mechanism of Brucella and influenza virus co-infection leading to acute lung injury and cytokine storm, screening characteristic indexes of co-infection acute lung injury and cytokine storm, and providing candidate targets for early diagnosis, prevention and clinical intervention treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of mouse model construction, and in particular to a Brucella-influenza A virus co-infection mouse model, a construction method and an application thereof. Background Art

[0002] In recent years, the ongoing global warming has dramatically altered the habitats of biomes. This, coupled with the deepening globalization process, may be one of the contributing factors to the recent global outbreaks of infectious diseases. Respiratory transmission is a major route of infectious disease transmission. The respiratory tract, directly connected to the outside world, serves as a gateway for numerous pathogens to invade the body. Against the backdrop of the current global epidemic of respiratory infections, we have found that the majority of patients with severe respiratory infections and deaths have co-infections with bacteria and viruses, as seen in diagnosis and treatment, as well as clinical mortality reports. Therefore, co-infection may be a significant factor in exacerbating the course of illness. Studies have shown that antibiotic treatment of patients infected with SARS-CoV-2 does not improve mortality or disease progression in patients with bacterial co-infections and may even increase mortality. This makes the treatment of patients with severe co-infections a difficult problem in clinical practice. Currently, for most acutely ill patients, the only options available are pulse corticosteroids and symptomatic treatment. Therefore, studying the excessive immune inflammatory response caused by bacterial-viral co-infection in the lungs from an immunological perspective can not only provide intervention plans to reduce the current clinical mortality rate of infectious pneumonia, but can also provide some diagnostic and treatment basis for unknown pandemics.

[0003] In recent years, the spread of Brucella has shown a resurgence. Brucella has high infection rates, high morbidity, a long incubation period after infection, is difficult to completely cure, and often leaves serious sequelae. Influenza A is the most widespread and pathogenic influenza virus subtype. It reproduces rapidly and often cross-infects multiple species, with the highest variability, often leading to local and global pandemics. To study the body's immune mechanisms against different pathogens and find effective prevention and control measures, it is necessary to establish an ideal animal model. Therefore, it is necessary to provide a Brucella-Influenza A virus co-infection mouse model and its construction method to facilitate the study of Brucella-Influenza A virus co-infection mechanisms and prevention and control measures. Summary of the Invention

[0004] The present invention aims to provide a Brucella-influenza A virus co-infection mouse model and a construction method thereof.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for constructing a Brucella-influenza A virus co-infection mouse model. After the mouse is anesthetized, the influenza A virus is infected by lung delivery. 10 to 14 hours later, the mouse is infected with Brucella by lung delivery.

[0007] Preferably, the influenza A virus is the influenza A H1N1 virus strain PR8.

[0008] Preferably, the Brucella is Brucella S2 strain.

[0009] Preferably, the inoculum size of the Brucella S2 strain is 10 7 CFU / piece.

[0010] Preferably, the influenza A (H1N1) virus strain is PR8 strain and the inoculation amount is 10 3 PFU / mouse.

[0011] The present invention also provides a Brucella-influenza A virus co-infection mouse model obtained according to the construction method.

[0012] The present invention also provides an application of the Brucella-influenza A virus co-infection mouse model in studying the pathogenesis and pathogenic mechanism of influenza virus and Brucella co-infection.

[0013] The Brucella-Influenza A virus co-infection mouse model provided by the present invention focuses on acute lung injury. In real environments, Brucella and influenza are commonly transmitted through aerosol infection, and aerosols are used as biological warfare agents due to their severe pathogenicity and high concealment. Therefore, the present invention uses Brucella vaccine strain S2 to calculate the appropriate challenge dose by LD50 and MID, with a maximum non-lethal dose of 10 7 CFU / mouse aerosol infection of C57BL / 6N mice. The acute phase lasted from day 2 to 4 after challenge, with pronounced respiratory and systemic symptoms. All mice harbored the bacteria in multiple organs. This indicates that this dose of S2 is highly virulent in mice and can be used to establish a co-infection model and subsequent mechanistic studies.

[0014] The virulence of influenza A virus PR8 was determined by LD50 and tissue TCID50, with a maximum non-lethal dose of 10 3C57BL / 6N mice were infected with PFU / mouse aerosol. On the third day after infection, the mice showed significant weight loss, with tissue TCID50 reaching 9.190E±2.074 / g, and symptoms such as huddling, piloerection, and shivering appeared. Thereafter, weight loss gradually decreased, and symptoms gradually worsened, with recovery occurring 7-8 days after infection. This indicates that this dose of PR8 is highly virulent in mice and can be used for the establishment of a co-infection model and subsequent mechanistic studies. The above strains and doses were then used with a 12-hour interval to establish a Brucella-influenza A co-infection model using lung delivery.

[0015] The Brucella-influenza A virus co-infection mouse model constructed in the present invention is relatively ideal and can be used to explore the synergistic pathogenic mechanism of acute lung injury and cytokine storm caused by co-infection of Brucella and influenza virus, screen characteristic indicators of co-infection acute lung injury and cytokine storm, and provide candidate targets for early diagnosis, prevention and clinical intervention treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Figure 2 is the weight change and survival curve of mice after challenge with the virus;

[0017] Figure 2 The pathological changes of lung tissues of mice on the third day after infection;

[0018] Figure 3 The bacterial and viral loads in the lungs were measured on the third day after challenge;

[0019] Figure 4 This is the change of various cytokines on the third day after the virus challenge;

[0020] Figure 5 is the relative expression level of NLRP6-related genes in the lungs of mice on the third day after challenge;

[0021] Figure 6 The bacterial / viral load and relative expression of pyroptosis-related genes in the lungs of mice on the third day after infection;

[0022] Figure 7 These are lung pathological sections of WT mice and gene knockout mice on the third day after infection. DETAILED DESCRIPTION

[0023] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1

[0025] 1. Materials

[0026] 1.1 Experimental Animals: SPF-grade 4-week-old female wild-type C57BL / 6N mice (WT, weighing 10–12 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., license number (SCXK (Beijing) 2016-0011).

[0027] 1.2 Cell Lines: The MDCK cell line used in this experiment was purchased from ATCC and provided by the cell bank of the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Academy of Military Sciences. MDCK cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% serum and 100 U / mL penicillin-streptomycin. Cells were passaged using trypsin, purchased from Gibco.

[0028] 1.3 Virus Strain: The influenza A (H1N1) virus strain used in this study was PR8 (A / PR / 8 / 1934), provided by the virus bank of the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Chinese Academy of Military Sciences. The H1N1 influenza virus was inoculated into 9- to 11-day-old SPF-free chicken embryos, amplified, and stored in a virus bank at -80°C in the laboratory. Virus titers were calculated using the median lethal dose (LD50) of MDCK cells (Reed-Muench method). All experiments involving viruses in this study were conducted in a biosafety level 2 laboratory.

[0029] 1.4 Bacteria strains: Brucella suis vaccine strain S2 was purchased from Xinjiang Tiankang Biological Co., Ltd.

[0030] 1.5 Main instruments and reagents

[0031] 96-well cell culture plates; chicken red blood cells; carbon dioxide incubator; electric constant-temperature water bath; low-temperature high-speed centrifuge; Trizol; 75% ethanol; RNase-free dH2O; NanoDrop 2000 nucleic acid concentration monitor; PCR instrument; fluorescence real-time quantitative PCR instrument; clean bench; refrigerator and ultralow-temperature freezer; electronic analytical balance; electric constant-temperature blast drying oven DGG-9023A; Tryptic Soy Agar medium; Tryptic Soy Broth medium; Brucella selective supplement; poloxamer 188 Sigma, USA; cytokine detection kit; small animal laryngoscope (Beijing Huironghe Technology Co., Ltd.); handheld liquid aerosol lung delivery device (Beijing Huironghe Technology Co., Ltd.); microplate reader; UV-visible spectrophotometer UV8000 Analytik Jena AG; tissue homogenizer ROCHE.

[0032] 2 Methods and Results

[0033] 2.1 Preparation of bacterial suspension: 20 μL of Brucella S2 glycerol culture was inoculated into 20 mL of TSB medium and cultured in a shaking incubator at 37°C at 200 rpm for about 16 h until the mid-logarithmic phase (OD 600nm ≈1.6), diluted 20-fold and transferred to 20 mL of TSB, continued to culture with shaking for about 8 h until the mid-logarithmic phase, centrifuged and collected the bacteria, washed twice with 0.05% poloxamer saline and resuspended again, and the OD was adjusted. 600nm The bacterial concentration was about 2×10 8 The CFU / mL (total bacterial concentration) is used to establish the mouse infection model in the later stage. A small amount of bacterial solution is taken for serial dilution and then plated for counting to calculate the actual bacterial concentration (viable bacterial concentration).

[0034] 2.2 Establishment of infection model

[0035] 1) Experimental Grouping: Four groups of 10 mice were inoculated via pulmonary delivery: one group was inoculated with Brucella S2; one group was inoculated with influenza virus H1N1 PR8; one group was inoculated with both S2 and PR8; and one group was inoculated with PBS. All mice were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital. Subsequent challenge experiments were performed after quadriplegia and scleral reflexes disappeared.

[0036] 2) Challenge dose and challenge time: The interval between two challenges was 12 hours.

[0037] Mice in group P (PR8) were inoculated with 50 μL PBS and 50 μL PR8 (10 3 PFU);

[0038] Mice in group B (Brucella) were inoculated with 50 μL S2 (10 7 CFU) and 50 μL PBS;

[0039] Mice in group G (co-infection) were inoculated with 50 μL PR8 (10 3 PFU) and 50 μL S2 (10 7 CFU);

[0040] Group D (Control) was inoculated with 100 μL PBS.

[0041] 3) Experimental animal challenge: Place the anesthetized mice on a dedicated experimental table for the lung delivery device. With the help of tweezers, clamp the mouse tongue and pull it out of the mouth. Open the laryngoscope and extend it along the mouse's oropharynx. You can see the epiglottis opening and closing with breathing. When the epiglottis opens, use the prepared handheld aerosol lung delivery device to penetrate deep into the throat to reach the trachea and quickly push a buckle. Be careful to avoid causing the mouse to choke or suffocate to death. After the mouse is inoculated, place its head high to prevent the liquid from flowing back through the mouse's trachea and nasal cavity to ensure the accuracy of the inoculation dose. The mouse woke up about 3 hours after inoculation and was observed for death. During the experiment, the mice were given sufficient food and water and 12 hours of light every day. The weight changes and deaths of the mice were recorded every day until the 14th day after infection, and the symptoms (hair raising, difficulty breathing, forced abdominal breathing, slow response to touch or external stimulation) were also recorded.

[0042] Results: Compared with the blank control group, mice in the single PR8 infection group began to huddle together, erect hair, and shiver on the third day after infection. On the sixth day, some mice developed respiratory distress, forced abdominal breathing, and slow reaction. By the eighth day after infection, the mortality rate reached 10%, and the remaining surviving mice gradually recovered. Mice in the single S2 infection group showed clinical manifestations such as huddle together, erect hair, and shiver on the first day after infection, and most began to recover on the 4th to 5th day, with no mouse death. Mice in the co-infection group showed obvious clinical symptoms on the second day after infection. On the sixth day, half of the mice in the group died, and all the other mice showed symptoms of dying. On the eighth day after infection, the mortality rate reached 80%, and the weight of the remaining surviving mice gradually recovered (such as Figure 1 shown).

[0043] 2.3 Pathological examination of mouse tissues and organs The lungs were aseptically isolated on the third day after infection and placed in a 4% formaldehyde solution for fixation for 48 h. The fixed samples were sent to the pathology laboratory for HE staining or fluorescent staining, and photographed using a dedicated microscope in the pathology laboratory.

[0044] Results: On the third day after co-infection, the mice were killed by removing their eyeballs, and the lung tissues of the mice were taken for pathological sections. It was found that compared with the control group, the co-infection group showed obvious alveolar wall rupture, alveolar fusion and bullae formation on the third day after infection. There were almost no alveoli with complete structure under the microscope, inflammatory cells infiltrated the alveolar walls, and the alveolar walls were obviously thickened. On the third day, the single PR8 infection group showed thickening and rupture of the alveolar walls, bullae formation, and inflammatory cell infiltration in the alveoli, but there were still a large number of alveoli with complete structures. On the sixth day, lung inflammation began to subside, and there was little infiltration in the alveoli, leaving the already formed bullae unable to recover. On the third day after infection, the single S2 infection group showed a large number of inflammatory cells infiltrating the alveolar walls and thickening, but only mild alveolar structural damage (such as Figure 2 shown).

[0045] 2.4 Colonization and detection of Brucella S2 in mouse lungs

[0046] On the third day after infection, 10 mice were selected from each group, and their lungs were aseptically isolated, weighed, homogenized, and diluted 5-fold into different concentrations. The bacterial solutions of different dilutions were spread on TSA medium supplemented with Brucella selective supplement. Three replicates were set for each dilution. The cells were cultured in an inverted manner at 37°C for 3-4 days and counted.

[0047] 2.5 Tissue TCID50 determination

[0048] The homogenized tissue suspension was filtered through a 0.45 μm filter and serially diluted 10-fold with serum-free medium. The suspension was then inoculated onto a 96-well microplate containing a monolayer of MDCK cells, with 100 μL added to each well. A blank control well was supplemented with 100 μL of medium, and each well was finally filled with medium to a total of 200 μL. The suspension was incubated at 37°C for 3-5 days, and the cytopathic effect was observed. On the fifth day of incubation, 50 μL of the suspension was aspirated from each well onto a hemagglutination plate, and PBS was added to the negative control and blank control wells. Subsequently, 50 μL of fresh chicken red blood cells was added to each well, and the cells were allowed to stand at room temperature for 15 minutes. The red blood cell aggregation was observed. The tissue TCID50 was determined based on the results of the hemagglutination test (Reed-Muench method).

[0049] 2.6 qRT-PCR detection of influenza virus RNA:

[0050] 1) 200 μL of mouse lung tissue homogenate + 300 μL of Trizol was immediately vortexed and allowed to stand for 5 minutes;

[0051] 2) Add 200 μL of chloroform, vortex for 15 seconds, let stand at 4°C for 5 minutes, and centrifuge at 12,000 rpm (13,400 g, 4°C) for 10 minutes;

[0052] 3) The sample has now separated into three layers: an upper aqueous phase, a middle buffy coat, and a lower organic phase. Carefully pipette 200 μL of the supernatant into a new RNase-free EP tube. Add 200 μL of isopropanol, gently invert to mix, and incubate on ice for 10 minutes (20-30 minutes at room temperature).

[0053] 4) Centrifuge at 12000 rpm (13400 g, 4°C) for 10 min and discard the supernatant;

[0054] 5) Add 1 mL of 75% ethanol (prepared with DEPC water), shake to mix, centrifuge at 7600 rpm (4°C) for 5 min, and discard the supernatant;

[0055] 6) Let the sediment dry at room temperature for 15 minutes (or around an alcohol lamp for 2-3 minutes);

[0056] 7) Add 20 μL RNase-free dH2O to dissolve the precipitate;

[0057] 8) Determine RNA concentration using a NanoDrop 2000 and perform reverse transcription using the following system and conditions:

[0058]

[0059] Reaction conditions: 25°C for 5 min, 42°C for 30 min, 85°C for 5 s, and 4°C for unlimited;

[0060] 9) Perform qPCR according to the following reaction system and conditions:

[0061]

[0062] Reaction conditions: 95°C for 10 min, 95°C for 15 s, 60°C for 60 s, 40 cycles.

[0063] Table 1 q-PCR primers:

[0064]

[0065] Results: The proliferation of the two pathogens was analyzed separately: on the third day after infection, the Brucella S2 plate count in the co-infection group was (7.974±0.2242) CFU / g, which was significantly higher than that in the single infection group (6.643±0.4695), and the difference was statistically significant (P<0.05). The TCID50 result of influenza virus PR8 in the co-infection group (6.490±0.3742) TCID50 / g and q-PCR result (7.673±0.9367) were significantly lower than those in the single infection group (9.190±0.8466) and q-PCR (13.70±2.589), and the difference was statistically significant (P<0.05). Figure 3 shown.

[0066] 2.7 Cytokine Detection

[0067] The homogenized lung tissue was centrifuged at 3000 rpm for 5 min at 4°C, and the supernatant was collected and stored at -20°C until further use. According to the instructions of the cytokine detection kit, the changes in cytokines (IFN-γ, TNF-α, IL-6, IL-1β, IL-18, IL-10, IP-10) in the lung tissue homogenate were measured on the third day after infection.

[0068] Results: The changes of cytokines on the third day after infection (such as Figure 4(As shown): Compared with the negative control group (5850±1096), the coinfection group had a significant increase in IFN-γ (10682±3088), with a statistically significant difference (P<0.05). However, compared with the single PR8 infection group and the single S2 infection group (10005±2557, 10198±1900), the coinfection group had a non-significant increase in TNF-α (6696±1593) compared with the negative control group, with a statistically significant difference (P<0.05). Compared with the single S2 infection group (1473±1663), the coinfection group had a significant increase in IL-6 (75891±1.525) compared with the negative control group (6665), with a statistically significant difference (P<0.05). Compared with the single PR8 infection group and the single S2 infection group (95529±16934, 44538±6522), the coinfection group showed no significant increase (P>0.05). Compared with the negative control group (416.0±295.2), the coinfection group showed a significant increase in IL-1β (731.6±126.1), with a statistically significant difference (P<0.05). However, compared with the single PR8 infection group and the single S2 infection group (578.7±331.8, 508.1±110.3), the coinfection group showed no significant increase (P>0.05). Compared with the negative control group (5850±1096), the coinfection group showed a significant increase in IL-18 (294715±124928), with a statistically significant difference (P<0.05). Compared with the single PR8 infection group and the single S2 infection group (101425±16307, 129342±55117), the coinfection group showed a significant increase (P>0.05). Compared with the negative control group (1622±696.6), the coinfection group showed a significant decrease in IL-10 (767.2±95.15), with a statistically significant difference (P<0.05). Compared with the single PR8 infection group and the single S2 infection group (2169±1358, 1042±253.6), the coinfection group also showed a significant decrease (P>0.05). Compared with the negative control group (7635±1659), the coinfection group showed a significant decrease in IP-10 (2775±179.1), with a statistically significant difference (P<0.05). Compared with the single PR8 infection group and the single S2 infection group (5261±1980, 4655±3554), the co-infection group was also significantly reduced, with statistical differences (P>0.05).

[0069] 2.8 Data Statistical Analysis

[0070] All data were analyzed by GraphPad Prism 8.0.2 software after statistics, and the data statistics were expressed as mean ± standard deviation, and one-way ANOVA was used between groups, and Kaplan-Meier survival analysis was used for animal survival rate. Significant difference is represented as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0071] As shown in Example 1, (1) compared with the two non-lethal models (single infection models), the mice in the co-infection group all showed obvious weight loss and respiratory and systemic symptoms on the second day after infection. On the sixth day after infection, the mice in the co-infection group showed a near-death state such as slow reaction, forced abdominal breathing, and hind limb paralysis, and a concentrated death trend appeared from 7 to 9 days after infection, with a mortality rate of 80-90%. (2) In order to explore the interaction between the two pathogens in mice in the early stage of the disease, the lung tissue of the mice was dissected and ground into homogenate on the third day after infection to measure the lung bacterial virus load. It was found that the Brucella load in the lungs of the mice in the co-infection group was significantly increased compared with the single infection group, and the difference was statistically significant, suggesting that influenza A virus infection promoted the proliferation and invasion of Brucella. However, in contrast, the influenza virus load in the co-infection group was significantly reduced compared with the single infection group, and the difference was statistically significant. This suggests that Brucella infection may inhibit the proliferation and invasion of influenza. Combined with the clinical manifestations of the mice, the massive proliferation of bacteria in co-infection may be the main factor leading to the poor prognosis of co-infection. (3) During the acute infection stage of Brucella, macrophages capture a large number of invading Brucella through endocytosis and clear a large number of captured Brucella through lysosomes and reactive oxygen species. However, recent studies have shown that intracellular parasitic pathogens such as Brucella, Chlamydia trachomatis, and coronavirus adopt different strategies to participate in the host cell autophagy pathway, and even evolve mechanisms to manipulate the expression and function of autophagy-related genes (ATG) to ensure the completion of their intracellular life cycle. Therefore, we speculate that due to influenza virus infection, the alveolar macrophage function of clearing pathogens is impaired and the non-classical autophagy pathway is upregulated, which may be the potential mechanism for the enhanced invasion and proliferation of Brucella in the co-infection group. Considering the impaired clearance function of macrophages and the severe clinical symptoms of mice in the co-infection group, it is considered that a strong excessive immune response and inflammatory response occurred in the early stage of co-infection. This excessive inflammatory immune response not only cannot effectively clear pathogens, but also causes immune damage to the body. When immune damage affects lung tissue, alveolar epithelial cells, as hosts of influenza viruses, may undergo premature pyroptosis or necrosis. Therefore, viral replication in alveolar epithelial cells is affected, and the viral load is lower than that in the single influenza infection group. (4) To clarify the immune status of infected mice and find the key points of immune imbalance, we measured a variety of representative cytokines related to viral and bacterial infections in mouse lung tissue. Compared with single infection, the expression of TNF-α, IL-18, IP-10 and IL-10 in co-infected mice was significantly different, suggesting that this type of cytokine may be the key factor leading to immune imbalance. IL-18R is widely expressed on various immune cells and binds to IL-18 to participate in the activation and pyroptosis of immune cells. Its abnormal increase often reflects the abnormal state of immune cells.This suggests that co-infected mice experienced a large amount of cell tissue necrosis and severe inflammatory response at the early stage of the disease. This conclusion is highly consistent with the previous clinical symptoms and lung tissue pathology. (5) The IP-10 level in the single infection group increased significantly, but the increase in IP-10 in the co-infection group was even more significant, suggesting that the co-infection group recruited more immune cells. However, although more inflammatory cells and immune cells were recruited, these cells did not seem to play a normal role in clearing pathogens. An important piece of evidence is the change in IL-10. IL-10 is the most important immune regulatory factor in the body and is mainly produced by activated mononuclear macrophages and DCs. We found that compared with the blank group, the IL-10 level in the single virus group increased significantly, while the change in the single bacteria group was not significant. However, the IL-10 level in the co-infection group decreased significantly, which has not been observed in previous studies. However, it is undeniable that such a significant decrease in IL-10 suggests that it has an important pathogenic mechanism in co-infection. Combined with the IP-10 results, it suggests that although the number of macrophages that chemotaxis to the local area increases, their immune function may have been damaged and they are unable to play a normal immune clearance function. And its impact may be even more far-reaching, because IL-10 can not only negatively regulate the innate immune response, but also regulate the acquired immunity towards Th2. Impaired macrophage function is likely to affect the function of acquired immunity.

[0072] Example 2

[0073] 1 Materials and Methods

[0074] 1.1 A co-infection mouse model was constructed according to the method of Example 1.

[0075] 1.2 Single-cell sequencing library construction, sequencing and data analysis were completed by Shanghai Oeuge Biomedicals Co., Ltd. The raw data generated by high-throughput sequencing was analyzed using the official software CellRanger of 10x genomics, and then the data was further quality controlled and processed based on the preliminary quality control results of Cellranger using the Seurat software package. Linear dimension reduction analysis was performed on the gene expression using PCA (principal component), and the PCA results were visualized in two-dimensional space by tSNE (nonlinear dimension reduction). Based on the single-cell reference expression quantitative public data set, the correlation between the expression profile of the cell to be identified and the reference data set was calculated, and the cell type with high correlation in the reference data set was assigned to the cell to be identified, which to some extent eliminated the interference of human subjective factors. Marker gene identification was performed using the FindAllMarkers function in the Seurant package, and the genes up-regulated in each cell classification relative to other cell groups were found. These genes are the potential Marker genes of each cell classification. The identified Marker genes were visualized by Vlnplot and FeaturePlot functions. Significant difference genes were screened using the FindMarkers function in the Seurat package, and significant difference genes were screened according to the conditions of p value less than 0.05 and difference more than 1.5 times.

[0076] 2 Results

[0077] 2.1 Neutrophils and macrophages are the main participating cells in the pathogenic process of Brucella-influenza virus co-infection

[0078] After quality control, the number of cells was: 6518 in S2+PR8 group, 6348 in single S2 group, 7327 in single PR8 group, and 7645 in PBS group. After dimension reduction clustering, differential expression gene analysis and analysis of the percentage of various cells in the whole lung cells, it was found that:

[0079] PBS group: monocytes 21.51%, neutrophils 5.25%, macrophages 31.4%, DC 4.02%, CD4+ T cells 3.38%, B cells 2.38%, natural killer cells (NK) 1.49%, NKT cells 0.66%, vascular endothelial cells (BEC) 26.47%, lymphatic endothelial cells (LEC) 0.63%, fibroblasts 14.39%, alveolar type II epithelial cells 3.21%, non-ciliated epithelial cells (Clara) 2.04%, glial cells (Muller) 0.49%.

[0080] Single S2 group: monocytes 45.7%, neutrophils 29%, macrophages 1.01%, DCs 2.5%, CD4+T cells 2.04%, B cells 1.23%, NK cells 0.49%, NKT cells 0.77%, BECs 5.91%, LECs 0.03%, fibroblasts 7.1%, alveolar type II epithelial cells 1.43%, Clara cells 0.15%, and Muller cells 0.06%.

[0081] Single PR8 group: monocytes 38.18%, neutrophils 12.44%, macrophages 10.34%, DCs 6.13%, CD4+T cells 5.81%, B cells 1.41%, NK 1.69%, NKT cells 1.32%, BECs 19.94%, LECs 0.12%, fibroblasts 7.01%, alveolar type II epithelial cells 6.87%, Clara 0.6%, Muller 0.54%.

[0082] S2+PR8 group: monocytes 32.98%, neutrophils 40.1%, macrophages 0.6%, DCs 2.74%, CD4+T cells 1.67%, B cells 1.15%, NK cells 0.21%, NKT cells 0.52%, BECs 9.4%, LECs 0.03%, fibroblasts 7.27%, alveolar type II epithelial cells 2.84%, Clara cells 0.34%, and Muller cells 0.14%.

[0083] Compared with the PBS group, these significantly altered cells may be involved in the body's defenses against Brucella and influenza virus infection. Compared with the S2 and PR8 alone groups, the coinfection group showed the most significant changes in the ratio of neutrophils to macrophages: a significant increase in neutrophils and a significant decrease in macrophages. This suggests that neutrophils and macrophages are the primary cells involved in the lethal process of acute lung injury caused by coinfection.

[0084] 2.2 Neutrophil Subpopulation Analysis

[0085] Through tSNE analysis, the neutrophils involved were divided into three clusters, and the percentage of each cluster in the neutrophils was statistically analyzed. The results showed:

[0086] PBS group: cluster C2 accounted for 0.91%; cluster C7 accounted for 2.44%; cluster C8 accounted for 1.9%;

[0087] PR8 group: cluster C2 accounted for 4.63%; cluster C7 accounted for 4.5%; cluster C8 accounted for 3.31%;

[0088] S2 group: cluster C2 accounted for 12.95%; cluster C7 accounted for 7.27%; cluster C8 accounted for 8.78%;

[0089] In the S2+PR8 group, cluster C2 accounted for 17.41%, cluster C7 accounted for 11.54%, and cluster C8 accounted for 11.15%.

[0090] PR8 and S2 infection can significantly increase neutrophil clusters C2, C7, and C8, suggesting that neutrophils are involved in the anti-infective immunity and immunopathogenesis of PR8 and S2. The proportion of each neutrophil subtype in the co-infection group is almost equal to the sum of the proportions in the single infection group, suggesting that the additive effect of the two pathogens on neutrophils may be one of the mechanisms underlying the worsening of the disease in the co-infection group.

[0091] Pseudo-time series analysis revealed that, compared with the PBS group, cluster C2 in the PR8 group was more likely to be at the differentiation starting point, C7 was beyond the differentiation point, and C8 was near the differentiation point. The S2 group showed a similar trend to the PR8 group. The coinfection group had four additional abnormal differentiation points compared to the PR8 group, with clusters C2 and C8 located at these points, while C7 remained beyond the differentiation point. This suggests that coinfection with S2 may cause abnormal differentiation of C2 and C7 into pathological subtypes. Compared with the S2 group, the coinfection group did not develop abnormal differentiation points, suggesting that PR8 coinfection did not lead to abnormal neutrophil differentiation.

[0092] Further analysis of the top 10 differentially expressed genes revealed significant changes in Ccl4 and Ngp in both single-infection groups compared to the PBS group, with opposite trends, suggesting a potential inhibitory effect between the two. Combined with the analysis of immune status in the first section, both single-infection groups showed improvement in their condition in the later stages, suggesting that this dose of challenge induced a self-regulating and self-healing immune response, rather than leading to immune imbalance. Changes in these genes in neutrophils may be self-regulatory.

[0093] Compared with the two single-infection groups, the addition of influenza virus significantly altered Ifnd1, S100ag, and S100a11; the addition of Brucella significantly altered Ccrl2, Cs1dc5, and Camp. Both influenza and Brucella significantly altered Ccl3, Cxcl2, Ccl4, S100a6, S100a8, Retnlg, and Ngp. The trends for Ccl3, Cxcl2, and Ccl4 were similar, as were those for S100a6, S100a8, Retnlg, and Ngp. However, these two gene groups exhibited opposite trends, consistent with those observed in the single-infection groups. Combined with the analysis of immune status in the first section, the co-infection group exhibited a higher incidence of late-stage mortality, suggesting that these abnormally altered genes may contribute to immune imbalance in co-infection, immune damage caused by neutrophil overactivation, and abnormal neutrophil differentiation following this dose of challenge.

[0094] Combined with the cytokine results in Example 1, we focused on the gene expression of IL-18 and TNF-α, which showed the most significant changes, as well as key factors in related pathways, in order to infer the similarities and differences in signal pathway activation under different infection conditions. The results showed:

[0095] The change characteristics of the PR8 group: Caspase-1, 4, TNF-α and IL-18 showed similar change trends, while Caspase-9 and GSDME showed similar change trends, suggesting that after viral infection, Caspase-1, 4, TNF-α and IL-18 participate in the same activation pathway, and Caspase-9 and GSDME participate in the same activation pathway in the signaling pathway initiated by the body.

[0096] The change characteristics of the S2 group: IL-18 and IL-1β, GSDMD have similar trends, and TNF-α and Caspase-4 have similar trends, suggesting that IL-1β and GSDMD are involved in the same activation pathway as IL-18, and Caspase-4 is involved in the same activation pathway as TNF-α.

[0097] Changes in the S2+PR8 group: In the coinfection group, Caspase-9 and GSDME showed similar trends, as did TNF-α and Caspase-4, both of which exhibited characteristics characteristic of the PR8 group. However, the changes in GSDMD maintained those of the S2 group. This suggests that the expression of Caspase-9, GSDME, TNF-α, and Caspase-4 in coinfection exhibits characteristics characteristic of influenza virus infection, but the relationship between the changes in GSDMD and influenza remains to be further confirmed. Therefore, the infection of these two pathogens is not simply a superposition but involves deeper immune mechanisms. With the addition of Brucella, the coinfection group showed similar trends for TNF-α and Caspase-4, and for IL-18 and IL-1β, but the changes in GSDMD lost the characteristics characteristic of the S2 group. This further suggests that coinfection induces additional changes in GSDMD regulation that differ from those in the two single infection groups. Whether these changes contribute to pathogen resistance remains unknown.

[0098] In summary, it is speculated that Caspase-9 and GSDME in neutrophils of mouse lungs are closely related to influenza virus infection, IL-1β and IL-18 are closely related to Brucella infection, but GSDMD may have a certain relationship with influenza virus-Brucella co-infection.

[0099] 2.3 Macrophage Subpopulation Analysis

[0100] Through tSNE analysis, the neutrophils involved were divided into 5 clusters, and the percentage of each cluster in the neutrophils was statistically analyzed. The results showed:

[0101] In the PBS group, cluster C4 accounted for 22.63%; cluster C16 accounted for 4.07%; cluster C19 accounted for 2.84%; cluster C27 accounted for 1.2%; cluster C29 accounted for 0.66%;

[0102] In the PR8 group, cluster C4 accounted for 6.24%; cluster C16 accounted for 2.27%; cluster C19 accounted for 1.33%; cluster C27 accounted for 0.18%; cluster C29 accounted for 0.32%;

[0103] In the S2 group, cluster C4 accounted for 0.87%, cluster C16 accounted for 0.08%, cluster C19 accounted for 0.02%, cluster C27 accounted for 0.02%, and cluster C29 accounted for 0.02%.

[0104] In the S2+PR8 group, cluster C4 accounted for 0.48%; cluster C16 accounted for 0%; cluster C19 accounted for 0%; cluster C27 accounted for 0%; cluster C29 accounted for 0.12%;

[0105] It can be seen that infection with PR8 and S2 can lead to a significant decrease in macrophage clusters C4, C16, C19, C27, and C29, suggesting that macrophages may be involved in the anti-infection immunity and immune pathogenesis mechanisms of PR8 and S2. Compared with the PBS group, the proportion of macrophage subtypes in all three infection groups was significantly reduced, with the co-infection group having the lowest proportion of macrophages, indicating that both pathogens can cause a decrease in macrophages, and the further decrease in macrophages caused by co-infection may be one of the mechanisms for the worsening of the disease in the co-infection group.

[0106] Pseudo-time series analysis revealed no abnormal differentiation points in macrophages in the PR8 and S2 groups compared with the PBS group; only a decrease in the number of individual subtypes was observed. This was significantly reduced in the S2 group, but all subtypes were still detectable. Compared with the PR8 and S2 groups, macrophage counts in the co-infection group were extremely low, with only C4 and C29 cells detectable. Therefore, the utility of pseudo-time series analysis is limited.

[0107] Further analysis of the top 10 differentially expressed genes: Compared with the PBS group, Histh1b, Histh1h2ae, Histh1h2ap, Mki67, Stmn1, and Hmgb2 in the two single infection groups all changed significantly, with the same trend of change, suggesting that these common trend genes may be related to the participation of macrophages in the body's anti-infection and cell death patterns. Combined with the analysis of immune status in Example 1, the condition of mice in the two single infection groups improved in the later stages, suggesting that this dose of the virus induced an immune response that could be self-controlled and self-healing, rather than causing immune imbalance. The changes in these genes in macrophages may be self-controllable.

[0108] Compared with two single infection groups, influenza virus and Brucella all make Hbb-bs significant changes, and in single infection group, no obvious change, suggesting that this gene may be the key gene of coinfection pathogenic mechanism.Except Hbb-bs, influenza virus and Brucella can also respectively promote other 9 kinds of different genes to change significantly.In conjunction with the analysis of embodiment 1 to immune status, coinfection group mouse later stage is more dead, prompting this dosage after attacking poison, this gene may be involved in the immune imbalance that macrophage reduces in a large number and causes, macrophage reduces antigen presentation and reduces the specific immune response defect that causes, and the local inflammation and lethal immune damage that a large amount of pathological death of macrophage causes.

[0109] Combined with the cytokine results in Example 1, we focused on the gene expression of IL-18 and TNF-α, which showed the most significant changes, as well as key factors in related pathways, in order to infer the similarities and differences in signal pathway activation under different infection conditions. The results showed:

[0110] The change characteristics of the PR8 group: TNF-α, Caspase-4, GSDMD and IL-1β had similar change trends, and IL-18, Caspase-9 and GSDME had similar change trends, suggesting that after viral infection, TNF-α, Caspase-4, GSDMD and IL-1β are involved in the same activation pathway, and IL-18, Caspase-9 and GSDME are involved in the same activation pathway in the signaling pathway initiated by the body.

[0111] The change characteristics of the S2 group: Caspase-1, 3, GSDMD, and IL-1β showed similar trends, and TNF-α, GSDME, and IL-18 showed similar trends.

[0112] Changes in the S2+PR8 group: With the addition of influenza virus, the co-infection groups showed similar trends in GSDMD, IL-18, and TNF-α, and similar trends in IL-1β and Caspase-1. The changes seen with influenza infection disappeared, but the trends for IL-18 and TNF-α retained those of the S2 group. This suggests that the expression of these genes in macrophages in the co-infection group does not display characteristics characteristic of influenza infection. Furthermore, similar relationships between GSDMD and IL-18 were observed in both the PR8 and S2 groups, but these relationships were no longer present in the co-infection group. This complex pattern suggests that the infection of these two pathogens is not simply additive but involves deeper immune mechanisms. With the addition of Brucella, the co-infection group showed similar trends in TNF-α, IL-1β, GSDMD, and Caspase-1, 3, and 4, and similar trends in IL-18 and Caspase-9. Interestingly, the changes in GSDMD exhibited similar patterns to those seen in the PR8 group, further suggesting that co-infection induces additional changes in GSDMD regulation that are distinct from those in the two mono-infection groups. Whether these changes contribute to pathogen resistance remains unknown.

[0113] In summary, it is speculated that TNF-α, Caspase-4, and GSDMD in mouse lung macrophages are closely related to influenza virus infection, and Caspase-1, 3, and GSDMD are closely related to Brucella infection, but GSDMD may have a certain relationship with Brucella-influenza virus co-infection.

[0114] As shown in Example 2, transcriptome analysis of lung tissue from mice co-infected with Brucella and influenza virus using 10×Genomics single-cell transcriptome sequencing revealed that, among all lung tissue cells, neutrophils and macrophages showed the most significant changes in co-infection. This may contribute to the increased severity of pneumonia and mortality in co-infected mice.

[0115] By combining the results of Example 1, we defined the single infection group as a non-lethal mild pneumonia model, the co-infection group as a potentially lethal severe pneumonia model, and defined the expression of each differential gene in the single infection group mice as being within the controllable range, while the co-infection group was in the abnormal uncontrollable range.

[0116] Example 3

[0117] 1 Materials and Methods

[0118] SPF 4-week-old NLRP6- / - knockout C57BL / 6N mice, weighing 10-12 g, were purchased from CYAGEN Biotechnology Co., Ltd.

[0119] Table 1 q-PCR primers:

[0120]

[0121] The remaining materials and methods are the same as in Example 1.

[0122] 2 Results

[0123] 2.1 Influenza infection upregulates NLRP6-mediated pyroptosis, which is an important cause of death in co-infected mice

[0124] Pyroptosis is a novel form of programmed cell death that relies on inflammatory caspases and is driven by inflammasomes. Pyroptotic cells exhibit characteristics of both apoptosis and necrosis, primarily characterized by positive Annexin V staining, a shrunken nucleus, and pores in the cell membrane. This leads to cell swelling and rupture, the release of cellular contents, and the secretion of inflammatory cytokines, triggering an inflammatory response. Therefore, pyroptosis is also known as inflammatory necrosis. Previous findings from this study showed a significant increase in IL-1β and IL-18 in the lung cytokine profile of mice with acute lung injury caused by coinfection, suggesting that excessive pyroptosis may be a key feature of coinfection. NLRP6 is a newly discovered member of the NLR protein family and the only protein in this family proven to negatively regulate the body, making it a hot topic of research in recent years. NLRP6 plays a crucial role in the induction of the host's innate immune response to pathogen infection. NLRP6 exhibits distinctly opposing functions in response to different pathogens and even in different organs or cells.

[0125] Based on the single-cell sequencing results of Example 2, it is believed that GSDMD and its related molecules may play an important role in the lethal process of coinfection. GSDMD-mediated pyroptosis is an important antimicrobial immune defense mechanism discovered in recent years. When pyroptosis occurs, it can participate in the occurrence and development of various diseases. Therefore, this example investigates the role of NLRP6 and GSDMD in coinfection acute lung injury.

[0126] In this example, it was observed that NLRP6 was fully upregulated in co-infected WT mice, but NLRP6 was only upregulated to a limited extent in the single infection model. Brucella and influenza virus infection can both upregulate NLRP6 expression to a certain extent, but the increase in NLRP6 in the co-infection group was much higher than that in the single infection group. This gives us reason to believe that it is the overexpression of NLRP6 and related molecules that is one of the important reasons for the increased mortality rate of mice caused by co-infection. We then measured the NLRP6 downstream pyroptosis gene Gasdermin and the NLRP6 inflammasome ASC-related gene PYCARD, as shown in Figure 3. Figure 5 As shown in the figure, Gasdermin and PYCARD in the co-infection group were significantly increased compared with the other two single infection groups, suggesting that the co-infection group showed obvious cell pyroptosis in the early stage of infection.

[0127] NLRP6- / - mice resist Brucella infection but develop uncontrolled influenza infection

[0128] To further explore the function of NLRP6 in lung co-infection, we constructed NLRP6 - / - In the co-infection model of gene knockout mice, the proliferation ability of Brucella was significantly inhibited compared with WT mice, and NLRP6 - / - The mice developed few obvious symptoms of bacterial infection. However, influenza virus expressed NLRP6 - / - A large number of proliferations occurred in mice. - / - In the lungs of mice, the trend of viral proliferation was completely opposite to that in WT mice: WT and NLRP6 - / - The viral expression levels in the single infection group of mice were almost the same. This indicates that the loss of NLRP6 does not affect the proliferation of the early virus. - / - The viral load of mice in the co-infection group was significantly increased, while the viral load of WT mice in the co-infection group was significantly decreased, indicating that co-infection effectively promoted the increase of NLRP6 expression, and the increase of NLRP6 inhibited the replication of the virus in the body, suggesting that Brucella infection may enhance the body's antiviral immune response by upregulating NLRP6 to a certain extent. Figure 6 shown.

[0129] Lung pathological sections showed that NLRP6 in the co-infection group and the single Brucella group- / - Mice had only mild lesions, whereas the single influenza A infection group showed obvious alveolar fusion and tissue infiltration (e.g. Figure 7 shown).

[0130] As shown in Example 3, when Brucella and influenza A virus co-infect the mouse lungs, the main cause of the mouse's death is the overreaction of the immune system and immune imbalance caused by the co-infection, rather than the direct effect of the pathogen.

[0131] In the WT mouse co-infection model, it was observed that compared with the two single infection groups, the co-infection group had a significant upregulation of NLRP6, an increase in the bacterial load in the lungs, but a significant decrease in the viral load. After knocking out NLRP6, the bacterial load in the co-infection group was significantly reduced, while the viral load was significantly increased. This suggests that after being challenged by bacteria and viruses, the body may effectively control viral infection by upregulating NLRP6-related immune responses, but cannot control Brucella infection. In contrast, the decrease in NLRP6 expression effectively controls bacterial infection, but cannot control viral infection. Therefore, excessive increase or decrease in NLRP6 will lead to an imbalance in the immune system, causing the body to lose its ability to control invading pathogens.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing a Brucella-influenza A virus co-infection mouse model, characterized in that: After anesthesia, mice were infected with influenza A virus by pulmonary delivery. Ten to fourteen hours later, they were infected with Brucella by pulmonary delivery. The influenza A virus is the influenza A H1N1 virus strain PR8, and the inoculation amount is 10 3 PFU / unit; The Brucella is the Brucella S2 strain, and the inoculation amount is 10 7 CFU / unit; The mice were 4-week-old female wild-type C57BL / 6N mice of SPF grade.

2. Application of a Brucella-influenza A virus co-infection mouse model obtained by the construction method of claim 1 in studying the pathogenesis, pathogenic mechanism, and prevention and control products of influenza virus and Brucella co-infection.

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