A66 EFFECTS OF HYMENOLEPIS DIMINUTA EXTRACELLULAR VESICLES ON MOUSE MACROPHAGES AND THEIR MITOCHONDRIA

Abstract

Abstract Background In recent years, extracellular vesicles (EVs) have emerged as important means of intercellular communication and immune modulation between helminth and host. Earlier work revealed that excretory/secretory products from the rat tapeworm Hymenolepis diminuta (HES) have anti-inflammatory properties when applied to macrophages. In this study, we have investigated the immunomodulatory properties of H. diminuta EVs (HEVs) on macrophages. Aims 1. To isolate and characterize bacterial contaminant-free H. diminuta EVs (Hd-EVs). 2. To determine the effects of Hd-EVs on murine macrophage metabolism and LPS-evoked cytokine production. Methods Adult H. diminuta were cultured for 6h in serum-free RMPI and Hd-EVs isolated from the conditioned medium using an exoEasy kit. Hd-EV protein content was measured using a micro-BCA kit, and size and quantity were measured using Nanosight tracking analysis. Murine bone marrow-derived macrophages or the human macrophage THP-1 cell line were treated with LPS (10 ng/ml) ± a crude H. diminuta extract (HdE), HES, or Hd-EVs for 24h. TNF-α levels in the culture medium was measured by ELISA. Macrophage oxidative phosphorylation (OX.PHOS) and glycolysis were measured in real-time using an Agilent Seahorse XF Analyzer. Results Analysis of 14 Hd-EV samples revealed 4.1 X 109 - 5.48 X 1010 particles/mL, with Hd-EVs predominantly 100-350 nm in diameter and with a protein content of 64.9-181.6 ng/mL. HdE and HES reduced TNF-α production in LPS-activated macrophages by 39-76% (n=11), while Hd-EVs did not lower TNF-α production. Metabolic analysis suggests that HES and Hd-EVs can up-regulate macrophage glycolysis, while only Hd-EVs exposed macrophages showed increased OX.PHOS (n=6). Conclusions We have successfully isolated and begun to characterize EVs from adult H. diminuta. Despite the known abilities of HES, we unexpectedly noted that exposure to Hd-EVs did not result in a statistically significant decrease in macrophage TNF-α release. However, the increase in OX.PHOS macrophages treated with Hd-EVs is something to examine further. The next steps are to determine the contents and immunomodulatory activity of the Hd-EVs, thus advancing knowledge of how helminths can manipulate their hosts. Funding Agencies NSERC Clinical Practice