Pre-exposing RAW264.7 Macrophages to Microbial Product, PAM3CSK4
Janice Pang
Summit Middle School
Floor Location : J 080 H

Studies have shown that lactic acid bacteria, a gram-positive bacterium, are a very important microorganism in our body and the environment. Not only is that, but it may also provide enhancements to our immune system. Phagocytic activities inside the human body play a vital role in defending and shaping strong barriers against infectious diseases too. White blood cells that circulate our body play a big role in allowing many different derivatives of macrophages to be formed. These macrophages are one of the first barriers against antigens and they are part of the innate immunity of our body. In this particular study, the phagocytic activity of macrophages was compared with those previously exposed to a microbial product. Since gram positive bacteria may enhance the immune system of human beings, the lipoprotein component, PAM3CSK4, of gram-positive bacteria was used to pre-expose RAW264.7 macrophages. This process of pre-exposing RAW264.7 macrophages to the lipoprotein is to test whether or not it would affect the outcome of phagocytic activity if it were to encounter gram-negative bacteria in the future. If prior exposure to the lipoprotein component, PAM3CSK4, shows an increased phagocytic uptake of gram-negative bacteria, then it would be another alternative for defeating infections caused by gram-negative bacteria. To test this, a group of RAW264.7 macrophages were pre-exposed to the lipoprotein, PAM3CSK4, while another group of the RAW264.7 macrophages were not. After the pre-exposure to the PAM3CSK4 lipoprotein, lipopolysaccharide (LPS), a component of gram-negative bacteria, were added along with red fluorescent microsphere beads to simulate a gram-negative bacteria antigen. Additionally, red fluorescent microsphere beads were added without LPS, so that it can be compared against the groups with LPS if the activation of phagocytic was dependent on LPS. After three trials of this experiment, phagocytic activities were then counted using the ImagePro Plus 6.1 software. Results revealed that pre-exposure to lipoprotein, PAM3CSK4, does enhance phagocytic activity of RAW264.7 macrophages by about 40%. Phagocytic activity activation of the RAW264.7 macrophages was not dependent on lipopolysaccharide (LPS) either. Beads added without lipopolysaccharide (LPS) had only 21.5% phagocytic activity decrease compared to macrophages treated with beads and lipopolysaccharide (LPS). Those without lipopolysaccharide (LPS) added sometimes had even more bead uptake than those with lipopolysaccharide. Thus, this study shows that pre-exposure to lipoprotein, PAM3CSK4, increases phagocytic activity of RAW264.7 macrophages up to 40%. Therefore lipoprotein, PAM3CSK4, can be a possible microbial product that can treat infections caused by pathogenic gram-negative bacteria by improving the phagocytic uptake.