Michael Fuller and Dr. Gregory Burton, Microbiology
Over the last semester, I have been engaged in studying the ability of macrophages to pick up HIV from follicular dendritic cells (FDC) in the follicles of the lymph nodes. Research by Dr. Gregory F. Burton of Brigham Young University has shown that HIV virions held on the dendrite processes of follicular dendritic cells in immune complex form are still infectious, and can infect T cells. Research by Dr. Suzanne Gartner at Johns Hopkins University has shown homology between HIV isolated from the brain to HIV in the lymph nodes. Tying these data together, I hypothesized that macrophages are able to pick up HIV in the lymph nodes, and then transport it to other sites in the body, such as the brain. To test this hypothesis, I designed an experiment to show that macrophages could be infected by HIV trapped on FDC.
For testing my hypothesis, the first thing I did was to obtain an R5-tropic strain of HIV. R5- tropic refers to the preferential binding of the virus to the CCR5 coreceptor for viral entry. The CCR5 coreceptor is more prevalent on macrophages than on T cells, this being the reason that R5-tropism was previously known as M-tropic, or macrophage-tropic. This virus was propagated in human macrophages to obtain a sufficient amount for running further experiments.
The next step in the experiment was to inject 5 mice with pooled human serum against HIV. This serum contained antibody against the HIV, thus allowing it to form immune complexes with HIV, which could then be trapped on the FDC. The mice were then injected with the R5-tropic HIV. After approximately 1 week, the mice were humanely sacrificed, and the lymph nodes were harvested. The lymph nodes were then digested, and the FDC were then harvested using magnetic beads with surface markers specific for markers on the FDC. Concurrently, macrophages were harvested from human blood. Blood was drawn, and lymphocytes were separated on a Ficoll-Paque density gradient. The lymphocytes were then incubated in flasks to allow the macrophages to adhere. Finally, the FDC were cocultured with the macrophages and allowed to incubate.
After about 1 week, all cells in the flask were removed, and were then lysed so that the DNA could be harvested from the cells. After the DNA was harvested, PCR was performed using primers for both the gag and env HIV genes. The samples were then run on an agarose gel, and then transferred onto a membrane for doing a Southern Blot. The Southern Blot is used for detection of specific DNA sequences, and is highly specific. After performing the Southern Blot, the presence of the env gene was detected. This indicates that infection did occur. The infection could have only been in the macrophages, because FDC are not infected by HIV, and the FDC were murine, and HIV is specific to humans. Thus, the infection occurred in the human macrophages.
Through the presence of HIV DNA in the human macrophages, I was able to show that infectious HIV can be transferred from FDC to macrophages. This can have further implications in the transport of HIV to other areas of the body, such as the brain, because the macrophages are able to cross the blood-brain barrier.