Immunomodulatory Impact of Leishmania-Induced Macrophage Exosomes: A Comparative Proteomic and Functional Analysis

Abstract

Released by many eukaryotic cells, the exosomes are 40–100 nm vesicles shown to operate over the complex processes of cell-cell communication. Among the metazoan cell lineages known to generate exosomes is the mononuclear phagocyte lineage, a lineage that parasites such as Leishmania are known to subvert as host cells. We previously reported that mouse macrophage signaling and functions are modified once co-incubated with exoproteome of Leishmania promastigotes. Using mass spectrometry analysis, we were curious to further compare the content of purified exosomes released by the J774 mouse macrophage cell line exposed or not to either LPS or to stationary phase Leishmania mexicana promastigotes. Collectively, our analyses resulted in detection of 248 proteins, ∼50–80% of which were shared among the three sources studied. Using exponentially modified protein abundance index (emPAI) and network analyses, we found that the macrophage exosomes display unique signatures with respect to composition and abundance of many functional groups of proteins, such as plasma membrane-associated proteins, chaperones and metabolic enzymes. Moreover, for the first time, L. mexicana surface protease GP63 is shown to be present in exosomes released from J774 macrophages exposed to stationary phase promastigotes. We observed that macrophage exosomes are able to induce signaling molecules and transcription factors in naive macrophages. Finally, using qRT-PCR, we monitored modulation of expression of multiple immune-related genes within macrophages exposed to exosomes. We found all three groups of exosomes to induce expression of immune-related genes, the ones collected from macrophages exposed to L. mexicana sharing properties with exosomes collected from macrophage left unexposed to any agonist. Overall, our results allowed depicting that protein sorting into macrophage-derived exosomes depends upon the cell status and how such distinct protein sorting can in turn impact the functions of naive J774 cells.

Author Summary

Secreted vesicles, such as exosomes, are now considered as an important route of communication among eukaryotic cells. Depending on the donor cell source and protein content, these vesicles are expected to distinctly impact the recipient cell properties. Here, three groups of exosomes released by the mouse macrophage cell line J774 exposed or not - naive exosomes - to either Leishmania mexicana promastigotes or to LPS were compared through proteomic analysis. Also, their biological activities on naive J774 macrophages were tested. Regardless of the source, the three groups of exosomes shared 50–80% of their proteins, although their relative abundances differed, especially those associated with the plasma membrane. Post exposure to one out of the three groups of exosomes, naive J774 recipient macrophages were compared for their profile of immune transcripts. Of note, whether they were exposed to either naive exosomes or to L. mexicana- induced exosomes, the naive J774 macrophages shared similar immune transcriptional signatures, the latter being distinct from the ones displayed by the macrophages exposed to LPS-induced exosomes. These data are discussed within the context of the unique cross talk that accounts for the early establishment of an immunomodulatory parasite such as Leishmania in its mammalian host.