New method for discussing IAV infected platelet-neutrophil interaction based on dynamic monitoring of different cell culture systems

Abstract

The invention relates to the field of immunology and cell biology, in particular to a novel method for discussing IAV infected platelet-neutrophil interaction based on dynamic monitoring of different cell culture systems, which comprises the following steps: (1) constructing three cell culture systems: a pure cell culture system, a Transwell co-culture system and a mixed co-culture system; (2) respectively infecting platelets and / or neutrophils in the three cell culture systems by using IAV; (3) in a culture process, dynamically detecting a lactic dehydrogenase release level, a neutrophil Calcein-AM / PI staining condition, a cell death phenotypic change and an expression level of a key medium for mediating cell interaction; and (4) comparing the detection results of the three cell culture systems to evaluate the interaction condition between the platelets and the neutrophils. The method disclosed by the invention is economical and practical, simple and convenient to operate and higher in repeatability, and interaction between IAV infected platelets and neutrophils can be researched under the condition of relatively conforming to real physiological conditions.

Description

Technical Field

[0001] This invention relates to the fields of immunology and cell biology, and in particular to a novel method for exploring platelet-neutrophil interactions in IAV infection based on dynamic monitoring of different cell culture systems. Background Technology

[0002] Neutrophils, as the first line of defense in the innate immune system, are highly recruited to the lungs in the early stages of influenza A virus (IAV) infection and release inflammatory mediators to participate in the immune response. However, their immune function is dualistic. Excessive recruitment and infiltration of neutrophils, leading to the release of large amounts of inflammatory cytokines, reactive oxygen species (ROS), or neutrophil extracellular traps (NETs), can cause an excessive immune inflammatory response, resulting in pathological tissue damage. Platelets, as sentinels of the immune defense, internalize the virus and activate it during IAV infection, releasing cytokines, inflammatory mediators, and antimicrobial peptides. They can recruit neutrophils and indirectly induce neutrophil activation and NET release through direct contact or by releasing platelet factor 4, high-migrating histone 1, and complement component 3 (C3), thereby participating in the immune response. In mouse models of severe influenza virus infection, platelet recruitment to the lungs can be observed early on. A significant increase in platelet-neutrophil aggregates (PNAs) can be detected in peripheral blood and bronchoalveolar lavage fluid. PNAs may migrate to the lungs and cause immunopathological damage to lung tissue. Therefore, studying platelet-neutrophil interactions plays an important role in revealing the pathological mechanisms of severe influenza virus infection.

[0003] Cell-cell interactions (CCIs) are fundamental to many cellular life activities. These interactions primarily coordinate gene expression and drive cellular function through structural and functional proteins, small molecule compounds, extracellular matrix or exosomes, and extracellular vesicles. Based on this theory, most researchers study CCIs using the following experimental methods: 1. Analyzing various omics studies, such as transcriptomics, proteomics, or single-cell sequencing, to assess the expression levels of receptor proteins or genes, thereby generating new biological hypotheses, and then evaluating the reliability of these hypotheses experimentally; 2. Co-localizing molecules involved in cell interactions using techniques such as fluorescence in situ hybridization, proximity-driven chemical labeling, cell imaging, or immunoprecipitation; 3. Isolating, identifying, labeling, tracing, performing omics analysis, or extracting key mediators of known cell interactions, such as exosomes and mitochondria, and co-culturing them with corresponding cells.

[0004] These methods, such as omics analysis or surface plasmon resonance, suffer from high experimental costs, difficulty in reproducibility, long experimental cycles, and the need for specialized equipment or high operational complexity. Methods such as cell imaging and immunoprecipitation require specific detection indicators and are generally only applicable to the experimental validation stage after the key receptors mediating CCIs have been identified. They are rarely used in early CCI studies and are also costly. Furthermore, separating and extracting known key mediators of cell-cell interactions and adding them to the corresponding cell culture system for co-culture cannot reflect the true interaction response of two cell types under normal physiological conditions at the same time and space.

[0005] Currently, there is no specific method for assessing cell interactions by dynamically monitoring three different cell culture systems. The methods used by various research institutions to study cell interactions generally suffer from high costs, long processing times, difficulty in reproducibility, or limited applicability in the early stages of cell interaction research. Therefore, developing a simple, cost-effective assessment method that better reflects normal physiological conditions is crucial for subsequent research on platelet-neutrophil interactions in IAV infection or on other cell interactions under other disease conditions. Summary of the Invention

[0006] The purpose of this invention is to provide a method that is simple to construct, economical, and more in line with normal physiological conditions to explore platelet-neutrophil interactions in IAV infection, addressing the problems mentioned above.

[0007] To achieve the above objectives, the present invention employs the following technical solution: a novel method for exploring platelet-neutrophil interaction in IAV infection based on dynamic monitoring of different cell culture systems, comprising the following steps:

[0008] (1) Three cell culture systems were constructed: a simple cell culture system, a Transwell co-culture system, and a mixed co-culture system; wherein, the simple cell culture system is a simple platelet culture system and a simple neutrophil culture system; the Transwell co-culture system is a platelet-neutrophil Transwell co-culture system, and in the Transwell co-culture system, the cell types in the upper and lower chambers can be adjusted; the mixed co-culture system is a platelet-neutrophil mixed co-culture system.

[0009] (2) Platelets and / or neutrophils in three cell culture systems were infected with IAV and then cultured at 37°C and 5% CO2.

[0010] (3) During the culture process, the release level of lactate dehydrogenase, the Calcein-AM / PI staining status of neutrophils, the changes in cell death phenotype, and the expression level of key mediators of interaction were dynamically detected in the three cell culture systems.

[0011] (4) By comparing the detection results of the three cell culture systems, assess whether there is an interaction between platelets and neutrophils, the type of interaction, and the biological effects of the interaction.

[0012] Preferably, in step (2), the IAV is a WSN virus strain with an infection multiplicity MOI of 0.5.

[0013] Preferably, in step (3), the dynamic detection is performed after 2 hours and / or 4 hours of incubation.

[0014] Preferably, in step (3), the changes in cell death phenotype are detected by immunofluorescence staining of three proteins: C-Caspase3, N-GSDMD, and P-MLKL.

[0015] Preferably, in step (3), the expression level of the key mediator of cell interaction is detected by enzyme-linked immunosorbent assay.

[0016] Preferably, the "assessment of the type of interaction" in step (4) includes: assessing indirect interactions mediated by cell secretion mediators by comparing the differences in detection results between the Transwell co-culture system and the simple culture system; and assessing direct interactions mediated by direct cell contact by comparing the differences in detection results between the mixed co-culture system and the Transwell co-culture system.

[0017] Preferably, the "evaluation of the biological effects of the interaction" in step (4) includes: evaluating whether the interaction accelerated the occurrence of the biological effect by comparing the time points of cell phenotypic changes in the Transwell co-culture system or mixed co-culture system with those in the simple culture system;

[0018] It also includes: by comparing the degree of phenotypic change of cells in the lower chamber at the same time point after adjusting the cell types in the upper and lower chambers in the Transwell co-culture system, to infer which cell type is the main driver of phenotypic changes in the entire culture system.

[0019] Preferably, the method further includes step (5): based on the potential key mediators identified in steps (3) and (4), adding recombinant proteins of the mediator to the simple cell culture system and monitoring changes in cell phenotypic indicators to verify whether the mediator mediates cell interactions and causes corresponding biological effects.

[0020] This invention also provides the application of the method in screening drugs that alleviate pathological damage caused by platelet-neutrophil interaction in IAV infection. Specifically, after constructing three culture systems, appropriate concentrations of the corresponding drugs are added to each system, and the release levels of lactate dehydrogenase, Calcein-AM / PI staining of neutrophils, and changes in cell death phenotypes are detected in the three systems. By comparing the changes in these indicators in different culture systems, the effectiveness of the drug in alleviating pathological damage caused by platelet-neutrophil interaction in IAV infection is assessed.

[0021] In addition, a method for studying cell interactions in other disease states is provided. Specifically, the method involves adjusting stimulation conditions and constructing three different culture systems for different cell types other than platelets and neutrophils to monitor changes in cell phenotypes other than cell death, thereby studying and exploring cell interactions in other disease states.

[0022] This invention also provides the application of the method in screening drugs that can mitigate pathological damage caused by cell interactions in other disease states. Specifically, three different culture systems are constructed for different cell types other than platelets and neutrophils. Appropriate concentrations of the corresponding drug are added to each of the three culture systems, and changes in cell phenotypes, such as lactate dehydrogenase release levels, neutrophil Calcein-AM / PI staining, or other cell types besides cell death, are detected in the three culture systems. Finally, by comparing the changes in these indicators in different culture systems, the effectiveness of the drug in mitigating pathological damage caused by cell interactions in other disease states is evaluated.

[0023] In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0024] The method provided by this invention for exploring platelet-neutrophil interactions in IAV-infected individuals is more economical, simpler to operate, and more reproducible. It can also study the interactions between platelets and neutrophils in IAV-infected individuals under relatively realistic physiological conditions, which is of great significance for early research on cell interactions between platelets and neutrophils in IAV-infected individuals.

[0025] The reagents and consumables used in this invention are easy to obtain and inexpensive, require no special or expensive equipment, are easy to operate, have high reproducibility, and skilled personnel can complete the construction of the culture system in a short time and quickly complete the sample detection. The experimental results are stable and reproducible.

[0026] The method provided by this invention can be used for early research on cell interactions: that is, by comparing the release levels of LDH in three different culture systems, it can assess whether there is an interaction between platelets and neutrophils, whether this interaction exacerbates inflammatory cell death, and explore the importance of different interaction modes.

[0027] The method provided by this invention can eliminate the temporal and spatial barriers in traditional co-culture conditions, and more realistically reflect the phenotypic changes caused by cell interactions under normal physiological conditions;

[0028] This invention allows for the adjustment of cell types in the lower chamber of a Transwell culture. By comparing the phenotypic changes of platelets and neutrophils in three different culture systems, cell interactions can be explored. For example, by comparing the phenotypic changes of the same cell at different time points in three different culture systems, cell interactions can be assessed to determine whether they exacerbate cell death. Alternatively, by comparing the phenotypic changes of different cells at the same time point in three different culture systems, cell interactions can be explored to determine which cell type might act as the driver of phenotypic changes in the entire culture system.

[0029] This invention explores the key mediators that mediate interactions by dynamically monitoring the expression of key mediators in different culture systems in real time: (1) by detecting the expression level of key mediators in a simple cell culture system to explore the cell origin; (2) by comparing the expression levels of key mediators in three different culture systems to explore the changes in mediators under interaction; (3) by adding recombinant proteins of key mediators or corresponding inhibitors to the corresponding culture systems and detecting changes in cell phenotypes to explore whether the mediators have the ability to mediate cell interactions and cause changes in cell phenotypes. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the method for exploring platelet-neutrophil interaction in IAV infection based on dynamic monitoring of three different cell culture systems.

[0031] Figure 2 This is a statistical graph showing the LDH release levels detected in three different cell culture systems in this invention.

[0032] Figure 3 The images show immunofluorescence patterns and statistical diagrams of neutrophils stained with Calcein-AM / PI under three different cell culture systems in this invention.

[0033] Figure 4 The images show immunofluorescence patterns and statistical graphs of C-Caspase3, N-GSDMD, and P-MLKL staining in platelets and neutrophils under three different cell culture systems in this invention.

[0034] Figure 5 This invention presents statistical graphs showing the secretion levels of potential key mediators in the interaction between IAV-infected platelets and neutrophils under three different cell culture systems, as well as statistical graphs showing the influence of key mediators on LDH release levels in platelets and neutrophils. Detailed Implementation

[0035] To more clearly illustrate the present invention, the following specific embodiments will be used to further explain the invention.

[0036] The schematic diagram of the method for exploring platelet-neutrophil interaction in IAV infection based on dynamic monitoring of three different cell culture systems is shown below. Figure 1 As shown.

[0037] The neutrophils and platelets used in this embodiment were obtained by extracting peripheral blood from healthy volunteers, and the specific steps are as follows:

[0038] (1) This project recruits healthy volunteers to collect blood for the following experiments. Selected volunteers must meet the following criteria: age 18-50 years, no history of hematologic diseases, no history of autoimmune diseases, no history of infectious diseases, no history of tumors, no history of immunosuppressive therapy, no history of infectious diseases in the past 2 weeks, and no history of taking anti-inflammatory, anticoagulant or antiplatelet drugs in the past 2 weeks.

[0039] (2) Platelet separation: Blood samples were collected and placed in a 5 mL blood collection tube containing 3.2% citrate. The blood samples were centrifuged at 180g for 10 minutes at room temperature to obtain platelet-rich plasma. Then, the platelet-rich plasma was centrifuged at 1800g for 10 minutes at room temperature to obtain platelet precipitate. The separated platelet precipitate was resuspended in preheated 10 mM HEPES modified 1640 medium. The cells were classified and counted using a hemocytometer to determine the platelet concentration and purity.

[0040] (3) Neutrophil isolation: Blood samples were collected and placed in 5 mL blood collection tubes containing EDTA anticoagulant. Then, 15 mL of 75% Percoll separation solution, 15 mL of 60% Percoll separation solution and whole blood were added sequentially. After centrifugation at 800 g density gradient for 30 min, the neutrophil layer (between 75% and 60% Percoll separation solution) was aspirated, washed with PBS, resuspended, and red blood cell lysis buffer was added to remove red blood cells. After washing twice with PBS again, the cells were resuspended in preheated 10 mM HEPES modified 1640 medium. Cell counts were performed to determine the concentration, and Wright staining was performed to determine the purity of neutrophils.

[0041] Example 1:

[0042] This embodiment provides a novel method for exploring platelet-neutrophil interactions in IAV-infected cells based on dynamic monitoring of different cell culture systems, including the following steps:

[0043] (1) Three cell culture systems were constructed: a simple cell culture system, a Transwell co-culture system, and a mixed co-culture system; wherein, the simple cell culture system was a simple platelet culture system and a simple neutrophil culture system; the Transwell co-culture system was a platelet-neutrophil Transwell co-culture system, and in the Transwell co-culture system, the cell types in the upper and lower chambers could be adjusted; the mixed co-culture system was a platelet-neutrophil mixed co-culture system; specifically, the different cell culture systems were constructed according to the following methods:

[0044] ① Construction method of platelet-only culture system: The extracted platelets were prepared with complete culture medium to a concentration of 1×10⁻⁶. 7 Cell suspension of 1 ml per cell was seeded into 12-well plates.

[0045] ② Construction method of simple neutrophil culture system: The extracted neutrophils were prepared with complete culture medium to a concentration of 1×10⁻⁶. 6 A cell suspension of 1 cell / ml was seeded into a 12-well plate, with 1 ml of cell suspension seeded into each well.

[0046] ③ Construction method of platelet-neutrophil Transwell co-culture system (platelets placed in the lower chamber of Transwell): The extracted platelets were prepared with complete culture medium to a concentration of 2×10⁻⁶. 7 Cell suspensions of 500 μL / ml were seeded into 12-well plates (i.e., the lower chamber of Transwell), with each well containing 500 μL of cell suspension. The extracted neutrophils were then reconstituted with complete culture medium to a concentration of 2 × 10⁻⁶ cells / ml. 6Cell suspension of cells / ml was seeded into Transwell chambers (also known as the upper Transwell chambers) and Transwell chambers were placed in 12-well plates, with 500 μL of cell suspension seeded into each well.

[0047] ④ Construction method of platelet-neutrophil Transwell co-culture system (neutrophils placed in the lower chamber of Transwell): The extracted neutrophils were prepared with complete culture medium to a concentration of 2×10⁻⁶. 6 Cell suspensions of 500 μL / ml were seeded into 12-well plates (i.e., the lower chamber of Transwell), with each well containing 500 μL of cell suspension. Platelets were extracted and reconstituted into a concentration of 2 × 10⁻⁶ cells / ml using complete culture medium. 7 Cell suspension of 500 μL / ml was seeded into Transwell chambers and the chambers were placed in 12-well plates, with each well seeded with 500 μL of cell suspension.

[0048] ⑤ Construction method of platelet-neutrophil co-culture system: Extracted platelets and neutrophils were prepared with complete culture medium to a platelet concentration of 2×10⁻⁶. 7 2 × 10⁶ cells / ml, neutrophil concentration 6 Cell suspensions of 500 μL / ml were seeded into 12-well plates, with 500 μL of platelet and neutrophil cell suspensions seeded into each well.

[0049] The composition of the above complete culture medium is: 86.5% 1640 medium, 10% 10 mM HEPES, 2.5% fetal bovine serum and 1% penicillin-dextrose antibody.

[0050] (2) After constructing the three culture systems, platelets and / or neutrophils in the three cell culture systems were infected with IAV WSN virus strain at MOI=0.5, and then cultured at 37℃ and 5% CO2. The culture time was then determined according to the differences in experimental groups.

[0051] (3) During the culture process, the release level of lactate dehydrogenase (LDH), neutrophil Calcein-AM / PI staining, changes in cell death phenotype, and expression levels of key mediators of the interaction were dynamically detected in the three cell culture systems described above; the specific detection methods are as follows:

[0052] ① Detect the LDH (lactate dehydrogenase) release level after IAV infection in different culture systems:

[0053] Three culture systems were constructed according to the above method (only one of the above types needs to be constructed for the platelet-neutrophil Transwell co-culture system). Platelets and neutrophils in different systems were infected with IAV WSN virus strain at MOI=0.5. Cell supernatants were collected from each culture system after 2 and 4 hours of culture. LDH enzyme activity in the cell supernatant was detected using a lactate dehydrogenase (LDH) kit. Finally, the chi-square test was used to analyze the differences between groups.

[0054] ② Detection of Calcein-AM / PI staining in neutrophils after IAV infection under three different culture systems.

[0055] The bottom of the culture dish was coated with poly-L-lysine beforehand, and three cell culture systems were constructed according to the above method. Platelets and neutrophils in different systems were infected with IAV WSN virus strain at MOI=0.5. After culturing for 4 hours, the cell supernatant was removed, and immunofluorescence staining was performed with Calcein-AM and PI dye at room temperature for 30 minutes. Finally, the staining was observed under a fluorescence microscope, and the results were statistically analyzed. The chi-square test was used to analyze the differences between the groups.

[0056] Esterases in living cells can catalyze the production of Calcein from Calcein-AM, which emits a strong green fluorescence. Calcein-AM can be used to stain and label living cells. PI, on the other hand, can penetrate the denatured regions of the cell membrane of dead cells to reach the cell nucleus, bind to DNA, and emit a red fluorescence, thus staining and labeling dead cells.

[0057] Since platelets do not contain a nucleus, Calcein-AM / PI fluorescence staining cannot reflect platelet cell death. Therefore, in simple cell culture systems, only a simple neutrophil culture system needs to be constructed, and there is no need to set up a simple platelet culture system. In platelet-neutrophil Transwell co-culture systems, only a platelet-neutrophil Transwell co-culture system with neutrophils placed in the lower chamber of the Transwell needs to be constructed, and there is no need to set up a platelet-neutrophil Transwell co-culture system with platelets placed in the lower chamber of the Transwell.

[0058] ③ Detection of cell death phenotypes in platelets and neutrophils after IAV infection under three different culture systems

[0059] a. After pre-constructing three culture systems using poly-L-lysine-coated cell slides, platelets and neutrophils in different systems were infected with IAV WSN virus strain at an MOI of 0.5. Cells from the neutrophil-only culture system, platelet-only culture system, platelet-neutrophil Transwell co-culture system (with neutrophils and platelets placed in the lower chamber respectively), and mixed co-culture system were incubated at 37°C and 5% CO2 for 2 and 4 hours, respectively. Then, the cell slides at the bottom of the culture dishes were removed.

[0060] b. Immunofluorescence staining: After removing the slides, fix them with 4% paraformaldehyde for 15 min, then rinse twice with PBS; block with 1% BSA for 1 h, then incubate with primary antibodies against C-Caspase3, N-GSDMD, and P-MLKL at room temperature for 1 h or overnight at 4°C, rinsing twice with PBS; then add the corresponding secondary antibodies and incubate at room temperature for 1 h, rinsing twice with PBS; add Alexa Fluro488, Alexa Fluro555, and Alexa Fluro640 and incubate in the dark for 10 min, rinsing twice with PBS. Next, add DAPI dye and incubate in the dark for 15 min, rinsing twice with PBS. Finally, mount the slides with an anti-fluorescence quencher and observe the staining using a confocal microscope to generate the corresponding fluorescence images.

[0061] Cleaved Caspase-3 (C-Caspase-3) is a highly specific and crucial molecular marker indicating apoptosis; N-terminal Gasdermin D (N-GSDMD) is a key executor in pyroptosis; and Phosphorylated Mixed Lineage Kinase domain-Like protein (P-MLKL) is a key executor in necroptosis. This experiment uses immunofluorescence staining of C-Caspase-3, N-GSDMD, and P-MLKL to indicate the type of cell death. DAPI dye was used to stain the nuclei of neutrophils to achieve neutrophil localization.

[0062] ④ Detect the release levels of key mediators in three different culture systems.

[0063] After constructing the three culture systems as described above, platelets and neutrophils in the different systems were infected with the IAV WSN virus strain at an MOI of 0.5. The neutrophil-only culture system, platelet-only culture system, platelet-neutrophil Transwell co-culture system, and platelet-neutrophil mixed co-culture system were incubated at 37°C and 5% CO2 for 2 and 4 hours, respectively. The cell culture supernatant was collected, and the key mediators of potential interactions were quantitatively detected using an ELISA (enzyme-linked immunosorbent assay) kit. Subsequently, the main sources of mediators were identified by comparing the simple culture systems, and the expression of key mediators under interaction conditions was identified by comparing the simple culture systems, Transwell co-culture system, and mixed co-culture system.

[0064] ⑤ The influence of key media on LDH release levels

[0065] By adding recombinant proteins of key mediators to a simple culture system and detecting LDH release levels after culture, we can explore whether this mediator mediates cell interactions and exacerbates cell death.

[0066] Example 2:

[0067] This embodiment explores the interaction between IAV-infected platelets and neutrophils based on specific experimental results, including the following:

[0068] 1. IAV-infected platelets interact with neutrophils and exacerbate cell death.

[0069] Three different culture systems were constructed, and platelets and neutrophils in the three systems were infected with the IAV WSN virus strain at a MOI of 0.5. LDH release levels were measured after 2 and 4 hours of culture at 37℃ and 5% CO2, respectively. It was observed that in the cell culture system alone, infection with the IAV WSN virus strain significantly increased LDH release levels in platelets and neutrophils. Compared with the infection group in the cell culture system alone, the infection groups in the Transwell co-culture system and the mixed co-culture system showed significantly upregulated LDH release levels (e.g., ...). Figure 2 As shown in AD), this indicates that IAV WSN virus strain infection of platelets and neutrophils interacts and mediates a significant upregulation of LDH release levels, meaning that this interaction exacerbates cell death. Furthermore, the significant upregulation of LDH release levels detected in the IAV WSN virus strain infection group in the Transwell co-culture system suggests that both indirect cell interactions mediated by cell secretion mediators and direct cell interactions mediated by direct cell contact play important roles in exacerbating platelet and neutrophil cell death between IAV WSN virus strain-infected platelets and neutrophils.

[0070] Subsequently, neutrophils cultured for 4 hours in three different culture systems were subjected to Calcein-AM / PI immunofluorescence staining. The results showed that in the neutrophil-only culture system, cell death was significantly upregulated in the IAV WSN virus strain infection group compared to the Control group; while compared to the infection group in the neutrophil-only culture system, cell death was significantly upregulated in the Transwell co-culture system and the mixed co-culture system (e.g., ...). Figure 3 (As shown in AB). The above results all indicate that platelet-neutrophil interaction caused by IAV WSN virus strain infection mediates accelerated cell death, and both direct and indirect interactions are of great significance.

[0071] 2. In the interaction between platelets and neutrophils in IAV-infected blood cells, platelets may act as a driver to accelerate the occurrence of panapoptosis.

[0072] Three different culture systems were constructed, and platelets and neutrophils in the three systems were infected with IAV WSN virus strain at a MOI of 0.5. Platelets in the platelet culture system alone and the lower chamber of the Transwell co-culture system were fixed and immunofluorescence staining for C-Caspase3, N-GSDMD, and P-MLKL was performed after 2 and 4 hours of culture. The results showed that compared with the control group in the platelet culture system alone, the IAV WSN virus infection group in the Transwell co-culture system showed a significant upregulation of C-Caspase3, N-GSDMD, and P-MLKL expression levels after 2 hours of culture (e.g., ...). Figure 4 (As shown in AD). Compared with the Control group in the platelet culture system alone, the IAVWSN virus strain infection group in the platelet culture system alone showed a significant upregulation of C-Caspase3, N-GSDMD, and P-MLKL expression levels after 4 hours of culture. This suggests that IAV WSN virus strain infected platelets interact with neutrophils and mediates and accelerates the occurrence of platelet PANoptosis, that is, PANoptosis, which usually occurs 4 hours after IAV WSN virus strain infected platelets, occurs as early as 2 hours.

[0073] Subsequently, neutrophils cultured for 4 hours in the simple neutrophil culture system, the lower chamber of the Transwell co-culture system, and the mixed co-culture system were fixed and subjected to immunofluorescence staining for C-Caspase3, N-GSDMD, P-MLKL, and DAPI. The results showed that compared with the Control group in the simple neutrophil culture system, only the N-GSDMD expression level was significantly increased in the IAV WSN virus strain infection group in the simple neutrophil culture system. However, in the Transwell co-culture system and the mixed co-culture system, the expression levels of C-Caspase3, N-GSDMD, and P-MLKL were simultaneously and significantly upregulated after 4 hours of culture (e.g., ...). Figure 4 As shown in EH), this suggests that platelet-neutrophil interaction mediated by IAV WSN virus strain infection accelerates neutrophil PANoptosis.

[0074] The above results indicate that platelet-neutrophil interaction mediated by IAV WSN virus strain infection accelerates cellular PANoptosis. In particular, PANoptosis, which usually occurs 4 hours after platelet infection in the Transwell co-culture system, occurs as early as 2 hours. The occurrence of PANoptosis by platelets may be accompanied by the release of a large amount of inflammatory mediators, suggesting that platelets may act as a driver to accelerate PANoptosis in the interaction between platelets and neutrophils infected by IAV WSN virus strain.

[0075] 3. IAV-infected platelets and neutrophils may interact through C3 secreted by platelets or IL-8 secreted by neutrophils, thus exacerbating cell death.

[0076] Finally, three different culture systems were constructed, and cells in all three systems were infected with IAV WSN virus strain at MOI=0.5 multiples. Cell supernatants were collected after 2 and 4 hours of culture, and the expression levels of key mediators potentially mediating platelet-neutrophil interactions were detected. The results showed that complement 3 (C3) was detected in the platelet-only culture system but not in the neutrophil-only culture system (e.g., ...). Figure 5 As shown in A); interleukin-8 (IL-8) was detected in neutrophil culture systems alone, but not in platelet culture systems alone (as shown in A). Figure 5 (As shown in B); this suggests that IL-8, which mediates the interaction, mainly originates from neutrophils, while C3 mainly originates from platelets.

[0077] After 2 hours of culture, the expression levels of C3 and IL-8 in the IAV WSN virus strain-infected group in the cell culture system were not significantly different from those in the control group; however, in both the Transwell co-culture system and the mixed co-culture system, the IAV WSN virus strain-infected group showed significant upregulation compared to the control group (e.g., Figure 5 (As shown in AB). The expression levels of C3 and IL-8 in the IAV WSN virus strain infection group in the Transwell co-culture system were significantly higher than those in the IAV WSN virus strain infection group in the simple culture system. This suggests that IAV WSN virus infection of platelets interacts with neutrophils and mediates the expression and secretion of C3 and IL-8. It also suggests that C3 and IL-8 may be key mediators of the interaction between IAV WSN virus infection of platelets and neutrophils. Specifically, the expression level of C3 in the IAV WSN virus strain infection group in the mixed co-culture system was significantly lower than that in the IAV WSN virus strain infection group in the Transwell co-culture system. This phenomenon may be due to: direct interaction between platelets and neutrophils exacerbating cell death, leading to a reduction in mediator production; or direct contact between platelets and neutrophils may have an inhibitory effect, inhibiting the synthesis and secretion of C3.

[0078] Finally, recombinant proteins IL-8 (50 μg / ml) and C3 (30 ng / ml) were added to platelet culture systems and neutrophil culture systems, respectively. LDH release levels were measured after culturing platelets for 2 hours or neutrophils for 4 hours. The results showed that adding C3 to the neutrophil culture system inhibited LDH release from neutrophils, while adding C3 to neutrophils infected with IAV WSN virus significantly increased LDH release levels (e.g., ...). Figure 5 (As shown in C); while the addition of IL-8 to platelet culture systems alone does not cause changes in LDH levels, the addition of IL-8 to platelets infected with IAV WSN virus strains significantly increases their LDH release levels (e.g., Figure 5 (As shown in D). The above results indicate that C3 and IL-8 may be key mediators of the indirect interaction between platelets and neutrophils in IAV WSN virus strain infection and the resulting exacerbation of cell death.

[0079] It should be noted that:

[0080] (1) The IAV WSN virus strain used in the embodiments of this application is: Influenza A virus strain A / WSN / 1933(H1N1), purchased from the American Type Culture Collection (ATCC);

[0081] (2) The Control group mentioned in the embodiments of this application refers to the control group that is not infected by the virus in a simple culture system; the Control group is different from the infected group in that it is not infected by IAV virus; the Control group is different from the infected group in Transwell co-culture and mixed co-culture in that it is not infected by IAV virus and does not interact with other cells.

[0082] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. A novel method for investigating platelet-neutrophil interaction in IAV-infected cells based on dynamic monitoring of different cell culture systems, characterized in that, Includes the following steps: (1) Three cell culture systems were constructed: a simple cell culture system, a Transwell co-culture system, and a mixed co-culture system; wherein, the simple cell culture system is a simple platelet culture system and a simple neutrophil culture system; the Transwell co-culture system is a platelet-neutrophil Transwell co-culture system, and in the Transwell co-culture system, the cell types in the upper and lower chambers can be adjusted; the mixed co-culture system is a platelet-neutrophil mixed co-culture system. (2) Platelets and / or neutrophils in three cell culture systems were infected with IAV and then cultured at 37°C and 5% CO2. (3) During the culture process, the release level of lactate dehydrogenase, the Calcein-AM / PI staining status of neutrophils, the changes in cell death phenotype, and the expression level of key mediators of cell interaction were dynamically detected in the three cell culture systems. (4) By comparing the detection results of the three cell culture systems, assess whether there is an interaction between platelets and neutrophils, the type of interaction, and the biological effects of the interaction.

2. The method according to claim 1, characterized in that, In step (3), the changes in cell death phenotype are detected by immunofluorescence staining of three proteins: C-Caspase3, N-GSDMD, and P-MLKL.

3. The method according to claim 1, characterized in that, In step (3), the expression level of the key mediators of cell-cell interaction is detected by enzyme-linked immunosorbent assay.

4. The method according to claim 1, characterized in that, The "assessment of the type of interaction" in step (4) includes: assessing indirect interactions mediated by cell secretion mediators by comparing the differences in detection results between the Transwell co-culture system and the simple culture system; and assessing direct interactions mediated by direct cell contact by comparing the differences in detection results between the mixed co-culture system and the Transwell co-culture system.

5. The method according to claim 1, characterized in that, The "evaluation of the biological effects of the interaction" in step (4) includes: evaluating whether the interaction accelerated the occurrence of the biological effects by comparing the time points of cell phenotypic changes in the Transwell co-culture system or mixed co-culture system with those in the simple culture system; It also includes: by comparing the degree of phenotypic change of cells in the lower chamber at the same time point after adjusting the cell types in the upper and lower chambers in the Transwell co-culture system, to infer which cell type is the main driver of phenotypic changes in the entire culture system.

6. The method according to claim 1, characterized in that, It also includes step (5): based on the potential key mediators identified in steps (3) and (4), adding the recombinant protein of the mediator to the simple cell culture system and monitoring changes in cell phenotypic indicators to verify whether the mediator mediates cell interactions and causes corresponding biological effects.

7. The use of the method according to any one of claims 1-7 in screening drugs that alleviate pathological damage caused by platelet-neutrophil interaction in IAV infection.

8. A method for studying cell interactions in other disease states, characterized in that, Using the method described in any one of claims 1 to 7, by adjusting the stimulation conditions and constructing three different culture systems for different cell types other than platelets and neutrophils, changes in cell phenotypes other than cell death are monitored to study and explore cell interactions in other disease states.

9. The use of the method according to any one of claims 1-7 in screening for drugs that alleviate pathological damage caused by cell interactions in other disease states.

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