A novel neuroinvasive infection modality for Francisella tularensis elicits neuroinflammation resulting in cellular damage

Francisella tularensis is a bacterium that causes a disease called Tularemia. To date, there is scarce information about the mechanisms the bacteria could use to infect the brain. However, previous research in our lab demonstrated that macrophages carry the most bacterial burden during infection. With this information coupled with the fact that the bacterium can escape the phagolysosome, led us to the exploration of the “Trojan Horse” mechanism used by F. tularensis to cross the blood brain barrier (BBB) and infect the brain. This project aims to elucidate this mechanism through a transcytosis assay and by analyzing the number of macrophages that cross the BBB in two groups: infected and uninfected macrophages. By using a 24 well plate and 8µm insert, Bend3 (endothelial mouse brain cells) cells were added to each group to mimic the BBB. To assess the integrity of the tight junctions in each well, for 10 days, the transepithelial/transendothelial electrical resistance of the Bend3 cells was measured with a Millicell Cell-Voltage meter. When the wells reached an average of 450 Ohms, infected macrophages and uninfected macrophages were added to their corresponding well. The macrophages used were Cdb11b and were extracted from C57BL/6 mice. We used confocal microscopy and fluorochromes to label the macrophages, bacteria, and Bend3 cells and imaged the passage of the macrophages. Through this, we found that more macrophages cross the BBB when infected.