Leishmania donovani infection of macrophages results in quantitative and qualitative changes in the protein profile of extracellular vesicles (EVs) released by the infected host cells. We confirmed mass spectrometry results orthogonally by performing Western blots for several Leishmania-infected macrophage-enriched EVs (LieEVs) molecules. Several host cell proteins in LieEVs have been implicated in promoting vascular changes in other systems. We also identified 59 parasite-derived proteins in LieEVs, including a putative L. donovani homolog of mammalian vasohibins (LdVash), which in mammals promotes angiogenesis. We developed a transgenic parasite that expressed an endogenously tagged LdVash/mNeonGreen (mNG) and confirmed that LdVash/mNG is indeed expressed in infected macrophages and in LieEVs. We further observed that LieEVs induce endothelial cells to release angiogenesis promoting mediators including IL-8, G-CSF/CSF-3, and VEGF-A. In addition, LieEVs induce epithelial cell migration and tube formation by endothelial cells in surrogate angiogenesis assays. Taken together, these studies show that Leishmania infection alters the composition of EVs from infected cells and suggest that LieEVs may play a role in the promotion of vascularization of Leishmania infections.

Extracellular vesicles (EVs) have garnered significant interest in recent years due to their contributions to cell-to-cell communication and disease processes. EVs are composed of a complex profile of bioactive molecules, which include lipids, nucleic acids, metabolites, and proteins. Although the biogenesis of EVs released by cells under various normal and abnormal conditions has been well-studied, there is incomplete knowledge about how infection influences EV biogenesis. EVs from infected cells contain specific molecules of both host and pathogen origin that may contribute to pathogenesis and the elicitation of the host immune response. Intracellular pathogens exhibit diverse lifestyles that undoubtedly dictate the mechanisms by which their molecules enter the cell’s exosome biogenesis schemes. We will discuss the current understanding of the mechanisms used during infection to traffic molecules from their vacuolar niche to host EVs by selected intravacuolar pathogens. We initially review general exosome biogenesis schemes and then discuss what is known about EV biogenesis in Mycobacterium, Plasmodium, Toxoplasma, and Leishmania infections, which are pathogens that reside within membrane delimited compartments in phagocytes at some time in their life cycle within mammalian hosts. The review includes discussion of the need for further studies into the biogenesis of EVs to better understand the contributions of these vesicles to host-pathogen interactions, and to uncover potential therapeutic targets to control these pathogens.

Although it is known that the composition of extracellular vesicles (EVs) is determined by the characteristics of the cell and its environment, the effects of intracellular infection on EV composition and functions are not well understood. We had previously shown that cultured macrophages infected with Leishmania parasites release EVs (LiEVs) containing parasite-derived molecules. In this study we show that LdVash, a molecule previously identified in LiEVs from L. donovani infected RAW264.7 macrophages, is widely distributed in the liver of L. donovani infected mice. This result shows for the first time that parasite molecules are released in EVs and distributed in infected tissues where they can be endocytosed by cells in the liver, including macrophages that significantly increase numbers as the infection progresses. To evaluate the potential impact of LiEVs on macrophage functions, we show that primary peritoneal exudate macrophages (PECs) express transcripts of signature molecules of M2 macrophages such as arginase 1, IL-10, and IL-4R when incubated with LiEVs. In comparative studies that illustrate how intracellular pathogens control the composition and functions of EVs released from macrophages, we show that EVs from RAW264.7 macrophages infected with Salmonella Typhimurium activate PECs to express transcripts of signature molecules of M1 macrophages such as iNOS, TNF alpha, and IFN-gamma and not M2 signature molecules. Finally, in contrast to the polarized responses observed in in vitro studies of macrophages, both M1 and M2 signature molecules are detected in L. donovani infected livers, although they exhibit differences in their spatial distribution in infected tissues. In conclusion, EVs produced by macrophages during Leishmania infection lead to the gene expression consistent with M2 polarization. In contrast, the EVs produced during S. Typhimurium infection stimulated the transcription of genes associated with M1 polarization.

Although it is known that the composition of EVs is determined by the characteristics of the cell and its environment, the effects of intracellular infection on EV composition and functions are not well understood. We had previously shown that cultured macrophages infected with Leishmania parasites release EVs that contain parasite derived molecules (LiEVs). In this study we show that LdVash, a molecule previously identified in LiEVs from L. donovani infected RAW264.7 macrophages is widely distributed in the liver of L. donovani infected mice. This result shows for the first time that parasite molecules are released in EVs and distributed in infected tissues where they can be endocytosed by cells in the liver, including macrophages that undergo a significant increase in numbers as the infection progresses. To commence evaluating the potential impact of LiEVs on macrophage functions, we show that primary peritoneal exudate macrophages (PECs) express transcripts of signature molecules of M2 macrophages such as arginase 1, IL-10 and IL-4R when incubated with LiEVs. In comparative studies that illustrate how intracellular pathogens control the composition and functions of EVs released from macrophages, we show that EVs from RAW264.7 macrophages infected with Salmonella enterica serovar Typhimurium activate PECs to express transcripts of signature molecules of M1 macrophages such as iNOS, TNF alpha and IFN gamma and not M2 signature molecules. Finally, we show that in contrast to the polarized responses observed in in vitro studies of macrophages, both M1 and M2 signature molecules are detected in L. donovani infected livers although they exhibit differences in their spatial distribution in infected tissues.