Exosomes Released during a Mycobacterium tuberculosis Infection Can Activate Endothelial Cells

Mycobacterium tuberculosis (M.tb), the causative agent of TB, is the leading cause of death due to an infection recently overtaking HIV in this dubious category. M.tb is an intracellular pathogen that primarily infects macrophages. The major focus of our laboratory is to understand how this intracellular pathogen interacts with the host immune system focusing primarily on exosomes, which are bioactive vesicles of 30-150nm in size that facilitate intercellular communication, and how they may be involved in modulating the host immune response. Although a significant effort has focused on characterizing host cell responses to antigen-loaded exosomes, we know little about how exosomes affect cellular function of the endothelium, which is an important regulator of host immune response during an M.tb infection. This dissertation aims to characterize the interaction between endothelial cells and exosomes derived from M.tb-infected macrophages. We also studied how exosomes isolated from the serum of M.tb-infected mice modulated endothelial cellular function ex-vivo.

Our previous studies indicate that exosomes released from M.tb-infected macrophages contain soluble mycobacterial proteins that can function in modulating the innate and acquired immune response. Interestingly, during a mouse M.tb or M.bovis BCG infection exosome concentration in serum correlated with bacterial burden. However, the function of these exosome is unclear. Endothelium, which can modulate immune responses by regulating leukocyte and antigen traffic, would be accessible and responsive to the exosomes in the serum. We hypothesize that the interactions between exosomes and endothelial cells would affect the immune response. To test this hypothesis, we treated the mouse endothelial cell line SVEC4-10 with exosomes released from uninfected or M.tb-infected macrophages. Our results indicate that endothelial cells treated with exosomes isolated from infected macrophages show increased permeability to dextran and enhanced macrophage migration through the endothelial cell monolayer. To evaluate the effects more globally, we sequenced the transcriptome of endothelial cells following exosomes treatment. We found 26 genes were significantly up-regulated in endothelial cells treated with exosomes derived from M.tb-infected macrophages compared to untreated cells, of which 12 were also up-regulated when compared to endothelial cells treated with exosomes derived from non-infected macrophages (q value < 0.05). Pathway analysis (using genes which show > 2-fold changes in expression) indicates that several immune response-related pathways were up-regulated in endothelial cells following exosome treatment.

Transcriptome studies defined a set of genes involved in adhesion and the inflammatory process were significantly up-regulated in endothelial cells treated with exosomes from M.tb-infected macrophages when compared to untreated cells or endothelial cells treated with exosomes from non-infected macrophages, including vcam1, tlr2 and ccl2. These results were validated by quantitative PCR. The upregulation of gene expression extended to protein level as flow cytometry data indicated enhanced expression of immune response-related proteins such as VCAM1, TLR2 and CCL2, when endothelial cells were treated with exosomes derived from M.tb-infected macrophages compared to cells treated with exosomes derived from non-infected macrophages. Moreover, immunofluorescence microscopy data indicated that the NF-kB pathway was activated in endothelial cells treated with exosomes released from M.tb-infected cells.

To test the function of the in vivo-derived exosomes, endothelial cells were also treated with exosomes isolated from the serum of M.tb-infected mice. Interestingly, exosomes isolated 14 days but not 7 or 21 days post-infection showed a similar ability to induce endothelial cell activation. We found that the same set of genes that were upregulated in our in vitro study were also increased at least 2 fold in endothelial cells when treated with exosomes derived from mice 14 days post M.tb infection. This differential expression extended to the protein level as CCL2 was expressed at the highest level when endothelial cells were treated with serum exosomes isolated 14 days post infection. Simillarly, there was enhanced macrophage migration through the monolayer when the endothelial cells were treated with these serum-derived exosomes. These results suggest a change in exosome composition and function during the course of an M.tb infection.

In summary, our data suggest that exosomes can activate endothelial cells and thus may play an important role in modulating the host immune response during a mycobacterial infection.