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.

Immunomodulatory components of helminths offer great promise as an entirely new class of biologics for the treatment of inflammatory diseases. Here, we discuss the emerging themes in helminth-driven immunomodulation in the context of therapeutic drug discovery. We broadly define the approaches that are currently applied by researchers to identify these helminth molecules, highlighting key areas of potential exploitation that have been mostly neglected thus far, notably small molecules. Finally, we propose that the investigation of immunomodulatory compounds will enable the translation of current and future research efforts into potential treatments for autoimmune and allergic diseases, while at the same time yielding new insights into the molecular interface of host-parasite biology.

Aconitum laciniatum is used in Bhutanese traditional medicine for treating various chronic infections and inflammatory conditions. We carried out in-depth isolation and characterization of the phytochemicals from the root component and determined the anti-inflammatory effects of the isolated compounds against chemically-induced colitis in mice. Five diterpenoid alkaloids - pseudaconitine, 14-veratroylpseudaconine, 14-O-acetylneoline, neoline, and senbusine A - were isolated from A. laciniatum for the first time. Two of the alkaloids were tested for anti-inflammatory properties in the TNBS-induced colitis model in mice. Various parameters were measured to assess pathology including weight loss, clinical and macroscopic scores, histological structure and IFN-γ production in the gut. Of the two alkaloids tested, 14-O-acetylneoline showed significant protection against different parameters of colitic inflammation. Compared to control mice that received TNBS alone, mice treated with 14-O-acetylneoline experienced significantly less weight loss and had significantly lower clinical scores, macroscopic pathology and grades of histological inflammation. Moreover, colonic IFN-γ mRNA levels were significantly reduced in mice that received 14-O-acetylneoline compared to control mice that received TNBS alone. This alkaloid is now considered a novel anti-colitis drug lead compound.

We present evidence that the dog hookworm (Ancylostoma caninum) is underutilised in the study of host-parasite interactions, particularly as a proxy for the human-hookworm relationship. The inability to passage hookworms through all life stages in vitro means that adult stage hookworms have to be harvested from the gut of their definitive hosts for ex vivo research. This makes study of the human-hookworm interface difficult for technical and ethical reasons. The historical association of humans, dogs and hookworms presents a unique triad of positive evolutionary pressure to drive the A. caninum-canine interaction to reflect that of the human-hookworm relationship. Here we discuss A. caninum as a proxy for human hookworm infection and situate this hookworm model within the current research agenda, including the various ‘omics’ applications and the search for next generation biologics to treat a plethora of human diseases. Historically, the dog hookworm has been well described on a physiological and biochemical level, with an increasing understanding of its role as a human zoonosis. With its similarity to human hookworm, the recent publications of hookworm genomes and other omics databases, as well as the ready availability of these parasites for ex vivo culture, the dog hookworm presents itself as a valuable tool for discovery and translational research.

No abstract

Hookworm infection is a major tropical parasitic disease affecting almost 500 million people worldwide. These soil-transmitted helminths can survive for many years in the intestine of the host, where they feed on blood, causing iron deficiency anemia and other complications. These parasites release a variety of molecules known as excretory/secretory products (ESPs) that are involved in many different biological processes that govern parasite survival. Using a combination of separation techniques such as SDS-PAGE and OFFGEL electrophoresis, in combination with state-of-the-art mass spectrometry we have reanalyzed the dog hookworm, Ancylostoma caninum, ESPs (AcESP). We identified 315 proteins present in the AcESP, compared with just 105 identified in previous studies. The most highly represented family of proteins is the SCP/TAPs (110 of the 315 proteins), and the most abundant constituents of AcESP are homologues of the tissue inhibitors of metalloproteases (TIMP) family. Interestingly, we identified new homologs of well-known vaccine candidates and immunomodulatory proteins. This study provides novel information about the proteins secreted by A. caninum, and constitutes a comprehensive dataset to study the proteins involved in host-hookworm interactions.

Hookworm infection is a major tropical parasitic disease affecting almost 500 million people worldwide. These soil-transmitted helminths can survive for many years in the intestine of the host, where they feed on blood, causing iron deficiency anaemia and other complications. To avoid the host’s immune response the parasite releases excretory/secretory products (ESPs), a complex mixture of glycans, lipids and proteins that represent the major host-parasite interface. Using a combination of separation techniques such as SDS-PAGE and OFFGEL electrophoresis, in combination with state-of-the-art mass spectrometry we have reanalysed the dog hookworm, Ancylostoma caninum, ESPs (AcES). We identified 315 proteins present in the AcES, compared with just 105 identified in previous studies. The most highly represented family of proteins is the SCP/TAPs (90 of the 315 proteins), and the most abundant constituents of AcES are homologues of the tissue inhibitors of metalloproteases (TIMP) family. We identified putative vaccine candidates and proteins that could have immunomodulatory effects for treating inflammatory diseases. This study provides novel information about the proteins involved in host-hookworm interactions, and constitutes a comprehensive dataset for the development of vaccines and the discovery of new immunoregulatory biologics.

Iatrogenic hookworm therapy shows promise for treating disorders that result from 27 a dysregulated immune system, including inflammatory bowel disease (IBD). Here we use a 28 metabolomics approach to characterize the non-protein small molecule complement of 29 hookworms. Gas chromatography-mass spectrometry and liquid chromatography-mass 30 spectrometry analyses of somatic tissue extracts revealed the presence of 52 polar metabolites 31 and 22 non-polar components including short chain fatty acids (SCFA). Several of these small 32 metabolites, notably the SCFA, have been shown to have anti-inflammatory properties in various 33 diseases, including IBD. Using a murine model of colitis and human peripheral blood 34 mononuclear cells, we demonstrate that somatic tissue extracts of the hookworm Ancylostoma 35 caninum contain small molecules with anti-inflammatory activities. Of the five extracts tested, 36 two of them significantly protected mice against T cell-mediated immunopathology and weight 37 loss in a chemically-induced colitis model. Moreover, one of the anti-colitic extracts suppressed 38 ex vivo production of inflammatory cytokines from primary human leukocytes. While the origin 39 of the SCFA (parasite or host microbiota-derived) present in the hookworm somatic tissue 40 extracts cannot be ascertained from this study, it is possible that A. caninum may be actively 41 promoting an anti-inflammatory host microbiome by facilitating immune crosstalk through SCFA 42 production. 43 44 Short chain fatty acids; GC-MS; Metabolome 46 47 48 49 50 3 51 52Inflammatory Bowel Disease (IBD) is associated with chronic inflammation of the 53 digestive tract, and primarily includes ulcerative colitis (UC) and Crohn's disease (CD). The 54 etiology of IBD is not well established but it is usually characterized by inflammation, loss of 55 appetite and weight, chronic diarrhea, bloody stools, fever, rectal bleeding, abdominal pain, 56 fatigue, and anemia (1-3). IBD has been linked to many extra-intestinal manifestations (4) and 57 implicated with mental health problems (5). Current treatments for IBD include, 5-58 aminosalicylates, glucocorticosteroids, immunomodulators and biologics, and proctocolectomy 59 as a last resort when drug treatment fails. Many of these drugs are often associated with side-60 effects and various postoperative complications (6, 7). Frontline biologics such as treatment with 61 anti-TNF monoclonal antibodies are only efficacious in some patients and treatment does not 62 result in long term cure (8). Failure of these frontline treatments is associated with elevated risk 63 of colon cancer, and can result in the need for surgical removal of the colon (partial or full). 64There is therefore an urgent need for new and effective anti-inflammatory drugs to treat IBD. 65Guided by millennia of host-parasite co-evolution, we (9-13) and others (14-16) have 66 demonstrated the therapeutic properties of experimental hookworm infection to treat 67 gastrointestinal (GI) inflammatory diseases. Hookworms resident in ...