| BackgroundLung infection is the most common respiratory diseases. In addition to direct damage, pathogens may also cause other diseases, for example, some studies indicated that the development of asthma may be related with S. aureus. The pathogens adhere to the mucus, and most are cleared via mucocilliary transport and via coughing. Representing the predominant species of lung phagocytes, airway and alveolar macrophages are able to remove the particles and pathogens deposited on the airway and alveolar surfaces. When the airways are infected by mounts of pathogens, neutrophils and macrophages are rapidly recruited to the sites of particle deposition to clear the pathogens. The recruited macrophages pass through the epithelium (basement membrane and epithelial cell layer) and arrive at the surface of airway. But there is no time-lapse image data to support the process of transmigration. To acquire the data of such cellular behaviors, the visualization of dynamic cellular processes in live animals is a top priority. However, acquiring pictures of cellular behaviors in a spatial and temporal manner is difficult in live animal models. Thus, in vitro cell culture models are of great importance. However, simple monocultures and static3D co-culture models in vitro are insufficient for transmigration studies. Thus, we combine3D airway infection model in vitro with time-lapse confocal imaging to investigate the transmigration and phagocytosis of recruited macrophages. Objectives1. To establish a three-dimensional co-culture model with multi-layers mimicking airway infection in vitro2. To study transmigration and phagocytosis of macrophages in an airway infection model by use of four-dimensional techniquesMethods1. Macrophage isolation and purificationThe macrophages were isolated by peritoneal lavage with6ml ice-cold PBS5days after an intraperitoneal injection of1ml of1.5%(w/v) starch solution. The cells were washed three times. We then removed the non-adherent cells by washing three times with PBS.2. Establishment of the airway infection modelLLC cells were seeded on the cell culture inserts formed an integral monolayer in5days. Then macrophages were added to the basal side of the inserts. At last, S.aureus was added to the upper chamber.3. TER measurementsLLC cells were seeded on the cell culture inserts. The TER was measured in the presence of S.aureus at different time points up to6hours.4. Transmigration of macrophages by time-lapse confocal imagingLLC cells were labeled with CellTracker Red after establishment of airway infection model. The inserts was moved into a glass-bottomed dish and images were taken in z-stacks and time-lapse using CLSM.5. Impact of S.aureus/MCP-1on transmigration of macrophages.Six hours after adding S.aureus/recombinant mouse MCP-1to the apical side of the inserts in the experimental groups, images were taken and transmigration index was evalued6. ELISA for MCP-1detectionLLC monolayers were apically stimulated with S.aureus for3hours. At the end of the incubation time, the apical and basal supernatants were harvested. The MCP-1levels of supernatants were quantified using ELISA kits following the manufacturer’s instructions.7. Apical to basal transmigration of macrophages by time-lapse confocal imagingLLC cells were seeded on the cell culture inserts. Five days later, the macrophages were plated on the apical surface and recombinant mouse MCP-1was add to the basal side of the inserts. The inserts was moved into a glass-bottomed dish and images were taken in z-stacks and time-lapse using CLSM.8. Macrophages phagocytosed S.aureus in airway infection model by time-lapse confocal imagingLLC cells were labeled with CellTracker Blue and S.aureus was labeled with Celltracker Red after establishment of airway infection model.The inserts was moved into a glass-bottomed dish and images were taken in z-stacks and time-lapse using CLSM.9. Statistical analysis.The SPSS13.0software was used for statistical analysis. Data were expressed as the means±SD. The height of macrophages was compared between groups using the one-tailed t’test. An independent t test was used to compare the groups with respect to the transmigration index and transmigration time. Differences were considered statistically significant if the P value was<0.05.Results1. Approximately5days after seeding, an integral monolayer of LLC cells had formed.Cells were seeded (0.5×106cells) on the cell culture inserts. The cell number steadily increased over time (from1061±97/mm2at24h to4954±256/mm2at120h) together with an increase in the percentage of cell area (27.3±6.0%at24h to99.8±0.3%at120h). Approximately5days after seeding, an integral monolayer of LLC cells had formed. 2. The integrity of LLC cell monolayer was not influenced by S.aureusBefore and after exposure to S.aureus, TER measurements were conducted over a6-hour period. When LLC cells monolayers were incubated with S.aureus (10/ml or108/ml), no change in TER was observed during the first6hours.3. Transmigration of macrophages by time-lapse confocal imaging3D reconstructions of the confocal data revealed that some of the macrophages transmigrated across the PET membrane to the apical aspect of the inserts through the pores in the PET membrane.4. S.aureus increased the transmigration of macrophages3D reconstructions of the confocal data revealed that some of the macrophages transmigrated across the PET membrane to the apical aspect of the inserts through the pores in the PET membrane.The transmigration index of macrophages in the models without S.aureus was significantly less than in the infection models5. S.aureus increased the secretion of MCP-1by LLC cellsThe MCP-1levels in the apical supernatants and basal supernatants of the models with or without S.aureus was detected by ELISA after LLC monolayers were apically stimulated with S.aureus for3hours. LLC cells secreted significantly more MCP-1in apical S.aureus supernatants than in apical non-S.aureus supernatants.6. Recombinant mouse MCP-lincreased the transmigration of macrophagesSix hours after incubation, transmigration index was also evalued and indicated that more macrophages transmigrated across the PET membrane to the apical aspect of the inserts in the model with recombinant mouse MCP-1.7. S.aureus influenced the movements of macrophages3D reconstructions of the confocal data revealed thatmacrophages on the apical side of the insert exhibited essentially three types of movements. S.aureus influenced the movements of macrophages.No macrophages transmigrated across the epithelial cell monolayer in the models without S.aureus8. Apical to basal transmigration of macrophages by time-lapse confocal imaging 3D reconstructions of the confocal data indicated that there were macrophages at the apical aspect transmigrated to the basal aspect. When the macrophages were plated on the apical surface and recombinant mouse MCP-1was add to the basal side of the inserts. Many macrophages were found transmigrating from the apical aspect to the basal aspect of the inserts.9. Phagocytosis of macrophages in airway infection model was time-dependent.3D reconstructions of the confocal data revealed that some S.aureus deposited on surface of monolayer attached to the macrophages which reached the surface of LLC monolayers and were then internalized by the macrophages.With time lapse, more and more S.aureus accumulated in the macrophages and more and more macrophages were found that had phagocytosed S.aureus.Conclusions1. Based on pathological structure of infected airway, we successfully established a3D airway infection model in vitro to study spatial behaviors of macrophages.2. The4D Techniques, which combined3D cell culture model in vitro with time-lapse confocal imaging, find its application in time-lapse imaging of spatial behaviors of cell, such as transmigration and phagocytizing S.aureus of macrophages in airway infection model. |
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