Influence Of Pseudomonas Aeruginosa Autoinducer Of Las On Murine Macrophage In Vitro

Pseudomonas aeruginosa is a versatile Gram-negative bacterium that grows in soil,marshes and coastal marine habitats ,as well as on plant and animal tissues. It is an opportunistic pathogen that causes a wide range of acute and chronic infections.However,the most prominent are the pulmonary infections.Due to the ubiquitous nature of Pseudomonas aeruginosa and its ability to develop resistance to many antibiotics, it has become the major cause of nosocomial pneumonia. These infections are impossible to eradicate, in part because of the natural resistance of the bacterium to antibiotics, and ultimately lead to pulmonary failure and death. This encouraged us working on the mechanism of infection so as to find an effective treatment.Recent study showed bacteria monitors the quantity change of itself or other bacterium in the surroundings according to concentration of special signal molecules. They adjust related genes to respond to such changes when signal shows concentration has reached certain threshold value. It is called Quorum-sensing system which universally exists in most of Gram-negative and Gram-positive bacteria. More researches of QS system showed it is relevant to many nosogenesis. The research of QS system of PA was very deep in Gram-negative pathogenic bacterium and we have known that QS system plays a key role in adjusting various virulence factors of PA. More researches on QS system confirmed that cell-to-cell signaling system is controlled by multiple genes and most of the genes are relevant to bacterial virulence. And also identified that the gene group which controlls cell-to-cell signaling system is a complicated cascade system. In this cascade system, except two main transcription regulation factors lasR and rhlR, it includes some other composition elements.Recent researches result demonstrated that QS system of PA, Las system is on the top of the cell-to-cell signaling cascade system. In cell-to-cell signaling system of PA, PQS is a hinge between Las system and Rh1 system. Las system is in the central. 3-oxo-C12-HSL will be produced in basal level when bacterial density is low, and if bacterial density increases, the density of 3-oxo-C12-HSL will rise up to the threshold value as well. At this time, 3-oxo-C12-HSL can combine with LasR, LasR-3-oxo-C12-HSL compound can control the network regulation and activate transcription of the number of lower genes. Accordingly, expression of a series of causative agents including pyocyanin can be activated too. Recently, it has become apparent that 3O-C12-HSL is not only important in the regulation of bacterial virulence genes but can also interact with eukaryotic cells and reduce an immune response. Recent progress has demonstrated the potential of quorum-sensing molecules, especially 3-oxo-C12-HSL,for inhibition of the host immune system. Also it may be an important regulate factor of the host immune response.The host have a better body's immune system. The immune system consists of immune organs, immune cells, as well as immune molecules. In the course of infection immunity, the immune organs, tissues, cells and immune molecules collaborate with each other and check each other closely together to complete complex function of the immune defense. After the bacteria invades the host, the first encounter is against the innate immune function. Innate immunity mainly consist of tissue barriers and certain immune cells, immune molecules and other components.While the macrophage as an important innate immune response cells which plays the role of secretion of cytokines, has the role of engulfing particulate antigen and the role of pinocytosis of soluble antigen. As the major phagocytic cells, macrophages are responsible for eliminating necrosised tissue, cell debris and pathogens at body injury in the phase of inflammation,Which have important roles of healing process .Therefore, the activation of macrophages of the early stage play a certain role in anti-infective immunity. So the behavior study of the change of apoptosis and phagocytosis of macrophages is new strategies for screening and development of new immunomodulatory drugs. We will provide preliminary evidence of further study of the interaction between bacteria and the host by observing the basic biological effects of chemically synthesized 3-oxo-C12-HSL molecule in the mouse macrophage cell line RAW264.7 cells.The studies were divided into two parts:Part one Construction, the detection of viability and apoptosis of Pseudomonas aeruginosa Autoinducer of Las on the mouse macrophage cell line RAW264.7 cell1. The detection of viability of Pseudomonas aeruginosa autoinducer of Las 3-oxo-C12-HSL on the mouse macrophage cell line RAW264.7 cellCell viability was evaluated by MTT assays . RAW264.7 cells were seeded into 96-well plates at a density of 8×106/ml, and cultured for overnight and then incubated in the presence or absence of 3-oxo-C12-HSL (100,50,25,12.5,6.25μM) at 37°C for 2 or 4 h in a humidified 5% CO2 and 95% air incubator(n=6). After treatment, 20μl of MTT solution (5 mg/ml in PBS) and 180μl of DMEM medium only were added to each well The viable cells were stained with MTT for 4 h. Media were removed and the formazan crystala was dissolved by adding 150μl of dimethylsulfoxide (DMSO). The optical density at 490nm was measured by a microplate reader to express the cell viabilities.2. The detection of apoptosis of Pseudomonas aeruginosa autoinducer of Las 3-oxo-C12-HSL on the mouse macrophage cell line RAW264.7 cell Assessment of apoptosis were seeded into 6 well plates at a right density and cultured for overnight and then incubated in in the presence or absence of 3-oxo-C12-HSL (100,50,25,12.5,6.25μM) at 37°C for 2 or 4 h in a humidified 5% CO2 and 95% air incubator. After treatment, Cells were measured by the binding of annexin V-FITC/PI using the protocol outlined in the Apoptest- FITC kit. Caspase 3,8 and 9activities were determined by colorimetric assays, in which caspase-specific peptides conjugated to the color reporter molecule p-nitroanilide were used. The data are expressed as fold increases compared to the values of control cells (n =3). JC-1 probe was employed to measure mitochondrial depolarization. We compare the apoptosis of RAW264.7 cells incubated in the presence or absence of 3-oxo-C12-HSL of different concentration through these three different assay methods.Part two Construction, the detection of Phagocytosis of Pseudomonas aeruginosa Autoinducer of Las on the mouse macrophage cell line RAW264.7 cell1.Phagocytic assay by neutral red method of Pseudomonas aeruginosa autoinducer of Las 3-oxo-C12-HSL on the mouse macrophage cell line RAW264.7 cellThe phagocytic ability of macrophage was measured by neutral red uptake. After cells were cultured with 3-oxo-C12-HSL or control (DMSO) for 2 h, 200μl neutral red solutions (dissolved in10 mM PBS with the concentration of 0.075%) was added and incubated for 1 h. The supernatant was discarded and the cells in 96-well plates were washed with PBS twice to remove the neutral red that was not phagocytized by RAW264.7 cells. Then cell lysate (ethanol and 0.01% acetic acid at the ratio of 1:1, 200μl /well) was added to lyse cells. After cells were incubated at 4°C overnight, the optical density at 490nm was measured by a microplate reader.2. Phagocytic assay by P. aeruginosa of Pseudomonas aeruginosa autoinducer of Las 3-oxo-C12-HSL on the mouse macrophage cell line RAW264.7 cell Phagocytosis experiments were performed using PAO1.Assessment of phagocytic ability were seeded into 6-well plates at a right density and cultured for overnight and then incubated in the presence or absence of 3-oxo-C12-HSL (100,50,25,12.5,6.25μM) at 37°C for 2 in a humidified 5% CO2 and 95% air incubator. After treatment, cells were incubated with Saline suspension of PAO1 for 1h(the number of Pseudomonas aeruginosa and cells is about 100:1), the supernatant was discarded and the cells in 6-well plates were washed with PBS three times to remove the PAO1 that was not phagocytized by RAW264.7 cells. phagocytic ratio and phagocytic index could be obtained by Wright staining observed in the oil Microscope. Conclusions:In this research different concentrations of 3-oxo-C12-HSL in mouse macrophage cells in culture and detection of various indicators of apoptosis and phagocytosis of change detection by oberserving the mouse macrophage cell line RAW264.7 cells's morphology, membrane changes , mitochondrial depolarization and Changes of the three key Caspase ,detection of changes in phagocytic capacity, indicated that Pseudomonas aeruginosa Las Signal molecule 3-oxo-C12-HSL cultured with the mouse macrophage cell line RAW264.7 cells have immunoinhibited activity and can reduce the macrophage immune response to infection.The results would provide primary evidence for further study in the interaction between bacteria and the host. If applyed in the clinical, it can be a new strategies for screening and development of new immunomodulatory drugs.