A11 isolation of non-polar metabolites in excretory/secretory products from parasitic helminths and their potential as immunotherapy in inflammatory bowel disease

Abstract Background Parasitic helminths are known to modulate host immune responses. This is thought to be mediated by their secretome. We are interested in the excretory/secretory products and mechanisms for modulating immune dysfunction in autoinflammatory diseases. Aims This research studies the mechanisms of immune modulation by parasitic helminths in the context of IBD. We aim to describe immunomodulatory helminth-derived metabolites (ESM). Methods Helminth-conditioned media was used to isolate ESM, which were further purified using column chromatography. Bone marrow (BM) derived macrophages (BMDM) from C57BL6 mice, were treated with ESP fractions from Trichuris suis, Ascaris suum, Heligmosomoides polygyrus bakeri or Dirofilaria immitis, stimulated with LPS, and secreted cytokine levels measured. Moreover, BM was cultured with or without ESM throughout differentiation to BMDM. Colitic mice (3% DSS, 5 days) were treated with A. suum ESM or PBS once daily IP. Colon lengths and TNFα mRNA were measured, and histological preparations were scored to assess pathology. Bioactive D. immitis ESM were fractionated using preparatory HPLC and assayed for bioactivity. Active fractions were analysed using MS/MS and fragmentation patterns and molecular weights were obtained. The active fractions are currently being studied by NMR to deduce a structure of an active metabolite. Results BMDM treated with crude ESM decreased TNFα secretion and increased IL-10. BMDM precursors which were treated with A. suum ESM throughout differentiation had reduced proliferation in a dose dependent manner. These BMDM showed remodeling of BMDM metabolic pathways. Intracellular ROS production was inversely proportional to Alamar blue oxidation. We found that ESM from A. suum improved DSS-colitis. Specifically, mice with DSS-induced colitis given IP ESM had longer colons, lower histolopathology score, and lower TNFα mRNA expression in gut tissue. HPLC-fractionated D. immitis ESM used to treat BMDM yielded varying suppression of TNFα with LPS stimulation. MS/MS of TNFα suppressive fractions contained masses with fragmentation patterns which were detected in fractions of several of the above-mentioned parasite species. Preliminary NMR studies will determine if this represents a conserved structure. Conclusions Helminth-derived components can immunologically polarize a response in vitro, as well as favour recovery in DSS colitis. Through multiple purification steps, a nearly pure fraction is found to have bioactivity, suggesting a single, bioactive molecule that is conserved across several parasitic helminths. These data are important in understanding the host-parasite interaction modulated by ESM, as well as provide therapeutic potential in IBD. Funding Agencies NSERC, FRQNT