Extracellular vesicles in host-parasite interaction of Entamoeba histolytica (Schaudinn, 1903) with primary monocytes

Abstract

Infection with the protozoan parasite Entamoeba histolytica (E. histolytica) is the cause of amebiasis. While most intestinal infections remain asymptomatic, a minority result in invasive disease. Invasive amebiasis, particularly amebic liver abscess (ALA) formation, occurs predominantly in adult men. An underlying immunopathology, crucially mediated by pro-inflammatory Ly6Chi monocytes, is known to contribute to liver damage in the murine model for the disease and differs between males and females. The mechanisms behind the switch from asymptomatic colonization to parasite invasion are incompletely understood to date. In order to investigate the interaction of the parasite with the host immune system, extracellular vesicles (EVs) as communicators between host and parasite were the focus of this study. EVs are membranous vesicles released by cells into their environment that contain protein, lipids, micro RNAs (miRNAs), and other types of cargo. They are involved in intercellular communication and pathogen-derived EVs have been demonstrated to modulate the host immune response in the context of a variety of infectious diseases. E. histolytica EVs and their effects on primary murine monocytes in vitro were studied here.

EVs were isolated from amebic clones of differing pathogenicity (low pathogenic A1 and highly pathogenic B2) and characterized with regard to their size, as well as protein and miRNA content. Mass spectrometry and miRNA sequencing revealed differences in the respective cargo between EVs of the two clones that may play a role in pathogenicity. Analysis of the proteome obtained by mass spectrometry showed that E. histolytica EVs contained known pathogenicity factors (such as galactose/N-acetylgalactosamine (Gal/GalNac) lectin and the cysteine peptidase (CP) EhCP-A5) and were enriched in transmembrane and signaling proteins. Furthermore, de novo miRNA prediction revealed novel mature E. histolytica miRNAs that were present in EVs. Stimulation of male and female primary murine monocytes with EVs in vitro resulted in a proinflammatory response independent of pathogenicity of the corresponding amebic clone. This was characterized by an increase in the secretion of pro-inflammatory cytokines TNFa, IL-1b, IL-6, and IL-12p40, as well as the chemokines CCL3, CXCL1, and CXCL10, which was partially ablated upon heat inactivation of B2 but not A1 EVs. In addition, EV-stimulated monocytes expressed higher levels of the activation marker CD38 on their surface compared with negative controls. Transcriptome analysis revealed that EV stimulation resulted in activation of key immune pathways, including TLR, TNF, and NF-kB signaling in both male and female monocytes. While the gene expression patterns induced by EV stimulation were similar between male and female monocytes, some sex-specific differences could be detected, for example in the expression of Lhfpl2. Stimulation with A1 EVs triggered increased secretion of the granular enzyme myeloperoxidase by monocytes, an effect not observed after B2 EV stimulation. The same was observed in peripheral neutrophils.

In summary, it was shown that A1 and B2 clones of E. histolytica release EVs containing immunogenic molecules that activate male and female monocytes, resulting in a pro-inflammatory phenotype.