Hookworm-Derived Metabolites Suppress Pathology in a Mouse Model of Colitis and Inhibit Secretion of Key Inflammatory Cytokines in Primary Human Leukocytes

Abstract

Iatrogenic hookworm therapy shows promise for treating disorders that result from a dysregulated immune system, including inflammatory bowel disease (IBD). Using a murine model of trinitrobenzenesulfonic acid-induced colitis and human peripheral blood mononuclear cells, we demonstrated that low-molecularweight metabolites derived from both somatic extracts (LMWM-SE) and excretorysecretory products (LMWM-ESP) of the hookworm, Ancylostoma caninum, display anti-inflammatory properties. Administration to mice of LMWM-ESP as well as sequentially extracted fractions of LMWM-SE using both methanol (SE-MeOH) and hexane-dichloromethane-acetonitrile (SE-HDA) resulted in significant protection against T cell-mediated immunopathology, clinical signs of colitis, and impaired histological colon architecture. To assess bioactivity in human cells, we stimulated primary human leukocytes with lipopolysaccharide in the presence of hookworm extracts and showed that SE-HDA suppressed ex vivo production of inflammatory cytokines. Gas chromatography-mass spectrometry (MS) and liquid chromatography-MS analyses revealed the presence of 46 polar metabolites, 22 fatty acids, and five short-chain fatty acids (SCFAs) in the LMWM-SE fraction and 29 polar metabolites, 13 fatty acids, and six SCFAs in the LMWM-ESP fraction. Several of these small metabolites, notably the SCFAs, have been previously reported to have anti-inflammatory properties in various disease settings, including IBD. This is the first report showing that hookworms secrete small molecules with both ex vivo and in vivo antiinflammatory bioactivity, and this warrants further exploration as a novel approach to the development of anti-inflammatory drugs inspired by coevolution of gutdwelling hookworms with their vertebrate hosts.

Excretory/Secretory Metabolome of the Zoonotic Roundworm Parasite Toxocara canis

“…This suggests that other diagnostic techniques should be considered, especially those that use more reliably measured chemical markers. Studies on other parasitic nematode ESPs have revealed the presence of diagnostically unique nucleic acids, glycans, lipids, and non-proteinaceous small biomolecules [25,55,56]. Therefore, characterising the metabolome of T. canis ESPs and identifying specific metabolites would be valuable for developing more sensitive diagnostics and biomarkers of infection, as well as potential therapeutics that exploit the immunoregulatory properties of helminth ESPs [57,58].…”

“…Sample derivatisation was performed as reported earlier for the GC-MS metabolomic analysis of ESPs derived from hookworms, whipworms and tapeworms [1,24,25]. The upper aqueous phase composed of 3:1 methanol:water (~900 µL) was aliquoted (50 µL) into a pulled point insert, dried using an evaporator at 30 • C (Christ RVC 2-33 CD, John Morris Scientific Australia) and then anhydrous methanol was added (50 µL).…”

“…The chloroform/organic layer of the ESPs and control samples was analysed via a GC-MS protocol described by us earlier [24,25]. In this protocol, the organic/chloroform phase of each biological replicate sample was sealed into GC vials and trans-esterified to generate fatty acid methyl esters.…”

“…Using previously described LC-MS protocols [24,25], we derivatised a small portion (20 µL) of the organic phase of the ESP extract (prepared using the 'Cryomill extraction method') using a 3-nitrophenylhydrazine (3-NPH), which converted the short chain fatty acids (SCFAs) to their 3-nitrophenylhydrazones. The samples (1 µL in triplicate) were then injected into a Shimadzu LC 30AD-TQ 8060 triple quadrupole mass spectrometer (SHIMADZU Scientific Instruments Inc., Columbia, MD, USA) connected to an Agilent EC-C18 poroshell 120 column (50 mm × 2.1 mm × 2.7 µm).…”

“…We found that seven non-polar MLCFAs including lauric acid (C12:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1, 9Z-cis), linoleic acid (C18:2), arachidonic acid (C20:4) and docosahexaenoic acid (C22:6) had been previously studied for their biological activities. Six MLCFAs (Table 2) and three SCFAs including acetate, propionate and butyrate were reported to possess anti-inflammatory properties [25]. While the most abundant saturated fatty acid, stearic acid, is known to possess anti-inflammatory activities; isovalerate, the major SCFA found in T. canis ESP had limited information on its biological properties.…”