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More than 470 million people globally are infected with the hookworms Ancylostoma ceylanicum and Necator americanus, resulting in an annual loss of 2.1 to 4 million disability-adjusted-life-years. Current infection management approaches are limited by modest drug efficacy, the costs associated with frequent mass drug administration campaigns, and the risk of reinfection and burgeoning drug resistance. Subunit vaccines based on proteins excreted and secreted (ES) by hookworms that reduce worm numbers and associated disease burden are a promising management strategy to overcome these limitations. However, studies on the ES proteomes of hookworms have mainly described proteins from the adult life stage which may preclude the opportunity to target the infective larva. Here, we employed high resolution mass spectrometry to identify 103 and 57 ES proteins from the infective third larvae stage (L3) as well as 106 and 512 ES proteins from the adult N. americanus and A. ceylanicum respectively. Comparisons between these developmental stages identified 91 and 41 proteins uniquely expressed in the L3 ES products of N. americanus and A. ceylanicum, respectively. We characterized these proteins based on functional annotation, KEGG pathway analysis, InterProScan signature and gene ontology. We also performed reciprocal BLAST analysis to identify orthologs across species for both the L3 and adult stages and identified five orthologous proteins in both life stages and 15 proteins that could be detected only in the L3 stage of both species. Last, we performed a three-way reciprocal BLAST on the L3 proteomes from both hookworm species together with a previously reported L3 proteome from the rodent hookworm Nippostrongylus brasiliensis, and identified eight L3 proteins that could be readily deployed for testing using well established rodent models. This novel characterization of L3 proteins and taxonomic conservation across hookworm species provides a raft of potential candidates for vaccine discovery for prevention of hookworm infection and disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Wong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Trichuris trichiura (whipworm) is one of the most prevalent soil transmitted helminths (STH) affecting 604-795 million people worldwide. Diagnostic tools that are affordable and rapid are required for detecting STH. Here, we assessed the performance of the near-infrared spectroscopy (NIRS) technique coupled with machine learning algorithms to detect Trichuris muris in faecal, blood, serum samples and non-invasively through the skin of mice., Methodology: We orally infected 10 mice with 30 T. muris eggs (low dose group), 10 mice with 200 eggs (high dose group) and 10 mice were used as the control group. Using the NIRS technique, we scanned faecal, serum, whole blood samples and mice non-invasively through their skin over a period of 6 weeks post infection. Using artificial neural networks (ANN) and spectra of faecal, serum, blood and non-invasive scans from one experiment, we developed 4 algorithms to differentiate infected from uninfected mice. These models were validated on mice from a second independent experiment., Principal Findings: NIRS and ANN differentiated mice into the three groups as early as 2 weeks post infection regardless of the sample used. These results correlated with those from concomitant serological and parasitological investigations., Significance: To our knowledge, this is the first study to demonstrate the potential of NIRS as a diagnostic tool for human STH infections. The technique could be further developed for large scale surveillance of soil transmitted helminths in human populations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kariyawasam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The reduced prevalence of insulin resistance and type 2 diabetes in countries with endemic parasitic worm infections suggests a protective role for worms against metabolic disorders, however clinical evidence has been non-existent. This 2-year randomised, double-blinded clinical trial in Australia of hookworm infection in 40 male and female adults at risk of type 2 diabetes assessed the safety and potential metabolic benefits of treatment with either 20 (n = 14) or 40 (n = 13) Necator americanus larvae (L3) or Placebo (n = 13) (Registration ACTRN12617000818336). Primary outcome was safety defined by adverse events and completion rate. Homoeostatic model assessment of insulin resistance, fasting blood glucose and body mass were key secondary outcomes. Adverse events were more frequent in hookworm-treated participants, where 44% experienced expected gastrointestinal symptoms, but completion rates were comparable to Placebo. Fasting glucose and insulin resistance were lowered in both hookworm-treated groups at 1 year, and body mass was reduced after L3-20 treatment at 2 years. This study suggests hookworm infection is safe in people at risk of type 2 diabetes and associated with improved insulin resistance, warranting further exploration of the benefits of hookworms on metabolic health., (© 2023. The Author(s).)

A decline in the prevalence of parasites such as hookworms appears to be correlated with the rise in non-communicable inflammatory conditions in people from high- and middle-income countries. This correlation has led to studies that have identified proteins produced by hookworms that can suppress inflammatory bowel disease (IBD) and asthma in animal models. Hookworms secrete a family of abundant netrin-domain containing proteins referred to as AIPs (Anti-Inflammatory Proteins), but there is no information on the structure-function relationships. Here we have applied a downsizing approach to the hookworm AIPs to derive peptides of 20 residues or less, some of which display anti-inflammatory effects when co-cultured with human peripheral blood mononuclear cells and oral therapeutic activity in a chemically induced mouse model of acute colitis. Our results indicate that a conserved helical region is responsible, at least in part, for the anti-inflammatory effects. This helical region has potential in the design of improved leads for treating IBD and possibly other inflammatory conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cobos, Bansal, Wilson, Jones, Zhao, Field, Eichenberger, Pickering, Ryan, Ratnatunga, Miles, Ruscher, Giacomin, Navarro, Loukas and Daly.)

Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host's immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm's excretory-secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen in vivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude in vitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases.

The symbiotic relationships shared between humans and their gastrointestinal parasites present opportunities to discover novel therapies for inflammatory diseases. A prime example of this phenomenon is the interaction of humans and roundworms such as the hookworm, Necator americanus. Epidemiological observations, animal studies and clinical trials using experimental human hookworm infection show that hookworms can suppress inflammation in a safe and well-tolerated way, and that the key to their immunomodulatory properties lies within their secreted proteome. Herein we describe the identification of 2 netrin domain-containing proteins from the N. americanus secretome, and explore their potential in treating intestinal inflammation in mouse models of ulcerative colitis. One of these proteins, subsequently named Na-AIP-1, was effective at suppressing disease when administered prophylactically in the acute TNBS-induced model of colitis. This protective effect was validated in the more robust CD4 T cell transfer model of chronic colitis, where prophylactic Na-AIP-1 reduced T-cell-dependent type-1 cytokine responses in the intestine and the associated intestinal pathology. Mechanistic studies revealed that depletion of CD11c+ cells abrogated the protective anticolitic effect of Na-AIP-1. Next generation sequencing of colon tissue in the T-cell transfer model of colitis revealed that Na-AIP-1 induced a transcriptomic profile associated with the downregulation of metabolic and signaling pathways involved in type-1 inflammation, notably TNF. Finally, co-culture of Na-AIP-1 with a human monocyte-derived M1 macrophage cell line resulted in significantly reduced secretion of TNF. Na-AIP-1 is now a candidate for clinical development as a novel therapeutic for the treatment of human inflammatory bowel diseases., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Coevolutionary adaptation between humans and helminths has developed a finely tuned balance between host immunity and chronic parasitism due to immunoregulation. Given that these reciprocal forces drive selection, experimental models of helminth infection are ideally suited for discovering how host protective immune responses adapt to the unique tissue niches inhabited by these large metazoan parasites. This review highlights the key discoveries in the immunology of helminth infection made over the last decade, from innate lymphoid cells to the emerging importance of neuroimmune connections. A particular emphasis is placed on the emerging areas within helminth immunology where the most growth is possible, including the advent of genetic manipulation of parasites to study immunology and the use of engineered T cells for therapeutic options. Lastly,we cover the status of human challenge trials with helminths as treatment for autoimmune disease, which taken together, stand to keep the study of parasitic worms at the forefront of immunology for years to come.

Competing Interests: PG is a co-founder of Paragen Bio, a biotech company that aims to translate and commercialise hookworm-derived proteins for inflammatory diseases. This has previously involved investment from pharma (Abbvie Inc) and venture capitalist firms (OneVentures, Brandon Capital). MS is founder and president of the biotech company NemaGen Discoveries. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Introduction: Celiac disease is an autoimmune disorder where intestinal immunopathology arises after gluten consumption. Previous studies suggested that hookworm infection restores gluten tolerance; however, these studies were small (n = 12) and not placebo controlled., Methods: We undertook a randomized, placebo-controlled trial of hookworm infection in 54 people with celiac disease. The 94-week study involved treatment with either 20 or 40 Necator americanus third-stage larvae (L3-20 or L3-40) or placebo, followed by escalating gluten consumption (50 mg/d for 12 weeks, 1 g intermittent twice weekly for 12 weeks, 2 g/d sustained for 6 weeks, liberal diet for 1 year)., Results: Successful study completion rates at week 42 (primary outcome) were similar in each group (placebo: 57%, L3-20: 37%, and L3-40: 44%; P = 0.61), however gluten-related adverse events were significantly reduced in hookworm-treated participants: Median (range) adverse events/participant were as follows: placebo, 4 (1-9); L3-20, 1 (0-9); and L3-40, 0 (0-3) (P = 0.019). Duodenal villous height:crypt depth deteriorated similarly compared with their enrolment values in each group (mean change [95% confidence interval]: placebo, -0.6 [-1.3 to 0.2]; L3-20, -0.5 [-0.8 to 0.2]; and L3-40, -1.1 [-1.8 to 0.4]; P = 0.12). A retrospective analysis revealed that 9 of the 40 L3-treated participants failed to establish hookworm infections. Although week 42 completion rates were similar in hookworm-positive vs hookworm-negative participants (48% vs 44%, P = 0.43), quality of life symptom scores were lower in hookworm-positive participants after intermittent gluten challenge (mean [95% confidence interval]: 38.9 [33.9-44] vs 45.9 [39.2-52.6])., Discussion: Hookworm infection does not restore tolerance to sustained moderate consumption of gluten (2 g/d) but was associated with improved symptom scores after intermittent consumption of lower, intermittent gluten doses.

Parasitic helminths have coevolved with humans over millennia, intricately refining and developing an array of mechanisms to suppress or skew the host's immune system, thereby promoting their long-term survival. Some helminths, such as hookworms, cause little to no overt pathology when present in modest numbers and may even confer benefits to their human host. To exploit this evolutionary phenomenon, clinical trials of human helminth infection have been established and assessed for safety and efficacy for a range of immune dysfunction diseases and have yielded mixed outcomes. Studies of live helminth therapy in mice and larger animals have convincingly shown that helminths and their excretory/secretory products possess anti-inflammatory drug-like properties and represent an untapped pharmacopeia. These anti-inflammatory moieties include extracellular vesicles, proteins, glycans, post-translational modifications, and various metabolites. Although the concept of helminth-inspired therapies holds promise, it also presents a challenge to the drug development community, which is generally unfamiliar with foreign biologics that do not behave like antibodies. Identification and characterization of helminth molecules and vesicles and the molecular pathways they target in the host present a unique opportunity to develop tailored drugs inspired by nature that are efficacious, safe, and have minimal immunogenicity. Even so, much work remains to mine and assess this out-of-the-box therapeutic modality. Industry-based organizations need to consider long-haul investments aimed at unraveling and exploiting unique and differentiated mechanisms of action as opposed to toe-dipping entries with an eye on rapid and profitable turnarounds., Competing Interests: The authors have declared that no competing interests exist.

Alternatively activated macrophages are essential effector cells during type 2 immunity and tissue repair following helminth infections. We previously showed that Ym1, an alternative activation marker, can drive innate IL-1R-dependent neutrophil recruitment during infection with the lung-migrating nematode, Nippostrongylus brasiliensis , suggesting a potential role for the inflammasome in the IL-1-mediated innate response to infection. Although inflammasome proteins such as NLRP3 have important proinflammatory functions in macrophages, their role during type 2 responses and repair are less defined. We therefore infected Nlrp3 -/- mice with N. brasiliensis Unexpectedly, compared with wild-type (WT) mice, infected Nlrp3 -/- mice had increased neutrophilia and eosinophilia, correlating with enhanced worm killing but at the expense of increased tissue damage and delayed lung repair. Transcriptional profiling showed that infected Nlrp3 -/- mice exhibited elevated type 2 gene expression compared with WT mice. Notably, inflammasome activation was not evident early postinfection with N. brasiliensis , and in contrast to Nlrp3 -/- mice, antihelminth responses were unaffected in caspase-1/11-deficient or WT mice treated with the NLRP3-specific inhibitor MCC950. Together these data suggest that NLRP3 has a role in constraining lung neutrophilia, helminth killing, and type 2 immune responses in an inflammasome-independent manner., (Copyright © 2019 The Authors.)

Helminth infections in children are associated with impaired cognitive development; however, the biological mechanisms for this remain unclear. Using a murine model of gastrointestinal helminth infection, we demonstrate that early-life exposure to helminths promotes local and systemic inflammatory responses and transient changes in the gastrointestinal microbiome. Behavioral and cognitive analyses performed 9-months postinfection revealed deficits in spatial recognition memory and an anxiety-like behavioral phenotype in worm-infected mice, which was associated with neuropathology and increased microglial activation within the brain. This study demonstrates a previously unrecognized mechanism through which helminth infections may influence cognitive function, via perturbations in the gut-immune-brain axis.

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite-host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis , which has been used as a model for human hookworm infection. N. brasiliensis EVs ( Nb -EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb -EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1β, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb -EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.

Inflammasomes promote immunity to microbial pathogens by regulating the function of IL-1-family cytokines such as IL-18 and IL-1β. However, the roles for inflammasomes during parasitic helminth infections remain unclear. We demonstrate that mice and humans infected with gastrointestinal nematodes display increased IL-18 secretion, which in Trichuris-infected or worm antigen-treated mice and in macrophages co-cultured with Trichuris antigens or exosome-like vesicles was dependent on the NLRP3 inflammasome. NLRP3-deficient mice displayed reduced pro-inflammatory type 1 cytokine responses and augmented protective type 2 immunity, which was reversed by IL-18 administration. NLRP3-dependent suppression of immunity partially required CD4 + cells but was apparent even in Rag1 -/- mice that lack adaptive immune cells, suggesting that NLRP3 influences both innate and adaptive immunity. These data highlight a role for NLRP3 in limiting protective immunity to helminths, suggesting that targeting the NLRP3 inflammasome may be an approach for limiting the disease burden associated with helminth infections., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Over 4.5 billion people are at risk of infection with soil transmitted helminths and there are concerns about the development of resistance to the handful of frontline nematocides in endemic populations. We investigated the anti-nematode efficacy of a series of polypyridylruthenium(II) complexes and showed they were active against L3 and adult stages of Trichuris muris, the rodent homologue of the causative agent of human trichuriasis, T. trichiura. One of the compounds, Rubb 12 -mono, which was among the most potent in its ability to kill L3 (IC 50  = 3.1 ± 0.4 μM) and adult (IC 50  = 5.2 ± 0.3 μM) stage worms was assessed for efficacy in a mouse model of trichuriasis by administering 3 consecutive daily oral doses of the drug 3 weeks post infection with the murine whipworm Trichuris muris. Mice treated with Rubb 12 -mono showed an average 66% reduction (P = 0.015) in faecal egg count over two independent trials. The drugs partially exerted their activity through inhibition of acetylcholinesterases, as worms treated in vitro and in vivo showed significant decreases in the activity of this class of enzymes. Our data show that ruthenium complexes are effective against T. muris, a model gastro-intestinal nematode and soil-transmitted helminth. Further, knowledge of the target of ruthenium drugs can facilitate modification of current compounds to identify analogues which are even more effective and selective against Trichuris and other helminths of human and veterinary importance., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Helminths have evolved to become experts at subverting immune surveillance. Through potent and persistent immune tempering, helminths can remain undetected in human tissues for decades. Redirecting the immunomodulating "talents" of helminths to treat inflammatory human diseases is receiving intensive interest. Here, we review therapies using live parasitic worms, worm secretions, and worm-derived synthetic molecules to treat autoimmune disease. We review helminth therapy in both mouse models and clinical trials and discuss what is known on mechanisms of action. We also highlight current progress in characterizing promising new immunomodulatory molecules found in excretory/secretory products of helminths and their potential use as immunotherapies for acute and chronic inflammatory diseases.

In the developed world, declining prevalence of some parasitic infections correlates with increased incidence of allergic and autoimmune disorders. Moreover, experimental human infection with some parasitic worms confers protection against inflammatory diseases in phase 2 clinical trials. Parasitic worms manipulate the immune system by secreting immunoregulatory molecules that offer promise as a novel therapeutic modality for inflammatory diseases. We identify a protein secreted by hookworms, anti-inflammatory protein-2 (AIP-2), that suppressed airway inflammation in a mouse model of asthma, reduced expression of costimulatory markers on human dendritic cells (DCs), and suppressed proliferation ex vivo of T cells from human subjects with house dust mite allergy. In mice, AIP-2 was primarily captured by mesenteric CD103 + DCs and suppression of airway inflammation was dependent on both DCs and Foxp3 + regulatory T cells (T regs ) that originated in the mesenteric lymph nodes (MLNs) and accumulated in distant mucosal sites. Transplantation of MLNs from AIP-2-treated mice into naïve hosts revealed a lymphoid tissue conditioning that promoted T reg induction and long-term maintenance. Our findings indicate that recombinant AIP-2 could serve as a novel curative therapeutic for allergic asthma and potentially other inflammatory diseases., (Copyright © 2016, American Association for the Advancement of Science.)

Schistosomiasis and trichuriasis are two of the most common neglected tropical diseases (NTD) that affect almost a billion people worldwide. There is only a limited number of effective drugs to combat these NTD. Medicinal plants are a viable source of parasiticides. In this study, we have investigated six of the 19 phytochemicals isolated from two Bhutanese medicinal plants, Corydalis crispa and Pleurospermum amabile, for their anthelmintic properties. We used the xWORM technique and Scanning Electron Microscope-based imaging to determine the activity of the compounds. Of the six compounds tested, isomyristicin and bergapten showed significant anthelmintic activity against Schistosoma mansoni and Trichuris muris with bergapten being the most efficacious compound one against both parasites (S. mansoni IC50 = 8.6 μg/mL and T. muris IC50 = 10.6 μg/mL) and also against the schistosomulum stage of S. mansoni. These two compounds induced tegumental damage to S. mansoni and affected the cuticle, bacillary bands and bacillary glands of T. muris. The efficacy against multiple phylogenetically distinct parasites and different life stages, especially the schistosomulum where praziquantel is ineffective, makes isomyristicin and bergapten novel scaffolds for broad-spectrum anthelmintic drug development that could be used for the control of helminths infecting humans and animals.

Background: Whipworms and blood flukes combined infect almost one billion people in developing countries. Only a handful of anthelmintic drugs are currently available to treat these infections effectively; there is therefore an urgent need for new generations of anthelmintic compounds. Medicinal plants have presented as a viable source of new parasiticides. Ajania nubigena, the Bhutanese daisy, has been used in Bhutanese traditional medicine for treating various diseases and our previous studies revealed that small molecules from this plant have antimalarial properties. Encouraged by these findings, we screened four major compounds isolated from A. nubigena for their anthelmintic properties., Methodology/principal Findings: Here we studied four major compounds derived from A. nubigena for their anthelmintic properties against the nematode whipworm Trichuris muris and the platyhelminth blood fluke Schistosoma mansoni using the xWORM assay technique. Of four compounds tested, two compounds-luteolin (3) and (3R,6R)-linalool oxide acetate (1)-showed dual anthelmintic activity against S. mansoni (IC50 range = 5.8-36.9 μg/mL) and T. muris (IC50 range = 9.7-20.4 μg/mL). Using scanning electron microscopy, we determined luteolin as the most efficacious compound against both parasites and additionally was found effective against the schistosomula, the infective stage of S. mansoni (IC50 = 13.3 μg/mL). Luteolin induced tegumental damage to S. mansoni and affected the cuticle, bacillary bands and bacillary glands of T. muris. Our in vivo assessment of luteolin (3) against T. muris infection at a single oral dosing of 100 mg/kg, despite being significantly (27.6%) better than the untreated control group, was markedly weaker than mebendazole (93.1%) in reducing the worm burden in mice., Conclusions/significance: Among the four compounds tested, luteolin demonstrated the best broad-spectrum activity against two different helminths-T. muris and S. mansoni-and was effective against juvenile schistosomes, the stage that is refractory to the current gold standard drug, praziquantel. Medicinal chemistry optimisation including cytotoxicity analysis, analogue development and structure-activity relationship studies are warranted and could lead to the identification of more potent chemical entities for the control of parasitic helminths of humans and animals.

Intestinal helminths are potent regulators of their host's immune system and can ameliorate inflammatory diseases such as allergic asthma. In the present study we have assessed whether this anti-inflammatory activity was purely intrinsic to helminths, or whether it also involved crosstalk with the local microbiota. We report that chronic infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb) altered the intestinal habitat, allowing increased short chain fatty acid (SCFA) production. Transfer of the Hpb-modified microbiota alone was sufficient to mediate protection against allergic asthma. The helminth-induced anti-inflammatory cytokine secretion and regulatory T cell suppressor activity that mediated the protection required the G protein-coupled receptor (GPR)-41. A similar alteration in the metabolic potential of intestinal bacterial communities was observed with diverse parasitic and host species, suggesting that this represents an evolutionary conserved mechanism of host-microbe-helminth interactions., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world.

Innate lymphoid cells (ILCs) are critical for maintaining epithelial barrier integrity at mucosal surfaces; however, the tissue-specific factors that regulate ILC responses remain poorly characterized. Using mice with intestinal epithelial cell (IEC)-specific deletions in either inhibitor of κB kinase (IKK)α or IKKβ, two critical regulators of NFκB activation, we demonstrate that IEC-intrinsic IKKα expression selectively regulates group 3 ILC (ILC3)-dependent antibacterial immunity in the intestine. Although IKKβ(ΔIEC) mice efficiently controlled Citrobacter rodentium infection, IKKα(ΔIEC) mice exhibited severe intestinal inflammation, increased bacterial dissemination to peripheral organs, and increased host mortality. Consistent with weakened innate immunity to C. rodentium, IKKα(ΔIEC) mice displayed impaired IL-22 production by RORγt(+) ILC3s, and therapeutic delivery of rIL-22 or transfer of sort-purified IL-22-competent ILCs from control mice could protect IKKα(ΔIEC) mice from C. rodentium-induced morbidity. Defective ILC3 responses in IKKα(ΔIEC) mice were associated with overproduction of thymic stromal lymphopoietin (TSLP) by IECs, which negatively regulated IL-22 production by ILC3s and impaired innate immunity to C. rodentium. IEC-intrinsic IKKα expression was similarly critical for regulation of intestinal inflammation after chemically induced intestinal damage and colitis. Collectively, these data identify a previously unrecognized role for epithelial cell-intrinsic IKKα expression and TSLP in regulating ILC3 responses required to maintain intestinal barrier immunity., (© 2015 Giacomin et al.)

Na-ASP-2 is an efficacious hookworm vaccine antigen. However, despite elucidation of its crystal structure and studies addressing its immunobiology, the function of Na-ASP-2 has remained elusive. We probed a 9000-protein human proteome microarray with Na-ASP-2 and showed binding to CD79A, a component of the B-cell antigen receptor complex. Na-ASP-2 bound to human B lymphocytes ex vivo and downregulated the transcription of approximately 1000 B-cell messenger RNAs (mRNAs), while only approximately 100 mRNAs were upregulated, compared with control-treated cells. The expression of a range of molecules was affected by Na-ASP-2, including factors involved in leukocyte transendothelial migration pathways and the B-cell signaling receptor pathway. Of note was the downregulated transcription of lyn and pi3k, molecules that are known to interact with CD79A and control B-cell receptor signaling processes. Together, these results highlight a previously unknown interaction between a hookworm-secreted protein and B cells, which has implications for helminth-driven immunomodulation and vaccine development. Further, the novel use of human protein microarrays to identify host-pathogen interactions, coupled with ex vivo binding studies and subsequent analyses of global gene expression in human host cells, demonstrates a new pipeline by which to explore the molecular basis of infectious diseases., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The development and severity of inflammatory bowel diseases and other chronic inflammatory conditions can be influenced by host genetic and environmental factors, including signals derived from commensal bacteria. However, the mechanisms that integrate these diverse cues remain undefined. Here we demonstrate that mice with an intestinal epithelial cell (IEC)-specific deletion of the epigenome-modifying enzyme histone deacetylase 3 (HDAC3(ΔIEC) mice) exhibited extensive dysregulation of IEC-intrinsic gene expression, including decreased basal expression of genes associated with antimicrobial defence. Critically, conventionally housed HDAC3(ΔIEC) mice demonstrated loss of Paneth cells, impaired IEC function and alterations in the composition of intestinal commensal bacteria. In addition, HDAC3(ΔIEC) mice showed significantly increased susceptibility to intestinal damage and inflammation, indicating that epithelial expression of HDAC3 has a central role in maintaining intestinal homeostasis. Re-derivation of HDAC3(ΔIEC) mice into germ-free conditions revealed that dysregulated IEC gene expression, Paneth cell homeostasis and intestinal barrier function were largely restored in the absence of commensal bacteria. Although the specific mechanisms through which IEC-intrinsic HDAC3 expression regulates these complex phenotypes remain to be determined, these data indicate that HDAC3 is a critical factor that integrates commensal-bacteria-derived signals to calibrate epithelial cell responses required to establish normal host-commensal relationships and maintain intestinal homeostasis.

Eosinophilic esophagitis (EoE) is a food allergy-associated inflammatory disease characterized by esophageal eosinophilia. Current management strategies for EoE are nonspecific, and thus there is a need to identify specific immunological pathways that could be targeted to treat this disease. EoE is associated with polymorphisms in the gene that encodes thymic stromal lymphopoietin (TSLP), a cytokine that promotes allergic inflammation, but how TSLP might contribute to EoE disease pathogenesis has been unclear. Here, we describe a new mouse model of EoE-like disease that developed independently of IgE, but was dependent on TSLP and basophils, as targeting TSLP or basophils during the sensitization phase limited disease. Notably, therapeutic TSLP neutralization or basophil depletion also ameliorated established EoE-like disease. In human subjects with EoE, we observed elevated TSLP expression and exaggerated basophil responses in esophageal biopsies, and a gain-of-function TSLP polymorphism was associated with increased basophil responses in patients with EoE. Together, these data suggest that the TSLP-basophil axis contributes to the pathogenesis of EoE and could be therapeutically targeted to treat this disease.

Resistin-like molecule (RELM)α belongs to a family of secreted mammalian proteins that have putative immunomodulatory functions. Recent studies have identified a pathogenic role for RELMα in chemically induced colitis through effects on innate cell populations. However, whether RELMα regulates intestinal adaptive immunity to enteric pathogens is unknown. In this study, we employed Citrobacter rodentium as a physiologic model of pathogenic Escherichia coli-induced diarrheal disease, colitis, and Th17 cell responses. In response to Citrobacter, RELMα expression was induced in intestinal epithelial cells, infiltrating macrophages, and eosinophils of the infected colons. Citrobacter-infected RELMα(-/-) mice exhibited reduced infection-induced intestinal inflammation, characterized by decreased leukocyte recruitment to the colons and reduced immune cell activation compared with wild-type (WT) mice. Interestingly, Citrobacter colonization and clearance were unaffected in RELMα(-/-) mice, suggesting that the immune stimulatory effects of RELMα following Citrobacter infection were pathologic rather than host-protective. Furthermore, infected RELMα(-/-) mice exhibited decreased CD4(+) T cell expression of the proinflammatory cytokine IL-17A. To directly test whether RELMα promoted Citrobacter-induced intestinal inflammation via IL-17A, infected WT and IL-17A(-/-) mice were treated with rRELMα. RELMα treatment of Citrobacter-infected WT mice exacerbated intestinal inflammation and IL-17A expression whereas IL-17A(-/-) mice were protected from RELMα-induced intestinal inflammation. Finally, infected RELMα(-/-) mice exhibited reduced levels of serum IL-23p19 compared with WT mice, and RELMα(-/-) peritoneal macrophages showed deficient IL-23p19 induction. Taken together, these data identify a proinflammatory role for RELMα in bacterial-induced colitis and suggest that the IL-23/Th17 axis is a critical mediator of RELMα-induced inflammation.

CD4(+) Th2 cytokine responses promote the development of allergic inflammation and are critical for immunity to parasitic helminth infection. Recent studies highlighted that basophils can promote Th2 cytokine-mediated inflammation and that phenotypic and functional heterogeneity exists between classical IL-3-elicited basophils and thymic stromal lymphopoietin (TSLP)-elicited basophils. However, whether distinct basophil populations develop after helminth infection and their relative contributions to anti-helminth immune responses remain to be defined. After Trichinella spiralis infection of mice, we show that basophil responses are rapidly induced in multiple tissue compartments, including intestinal-draining lymph nodes. Trichinella-induced basophil responses were IL-3-IL-3R independent but critically dependent on TSLP-TSLPR interactions. Selective depletion of basophils after Trichinella infection impaired infection-induced CD4(+) Th2 cytokine responses, suggesting that TSLP-dependent basophils augment Th2 cytokine responses after helminth infection. The identification and functional classification of TSLP-dependent basophils in a helminth infection model, coupled with their recently described role in promoting atopic dermatitis, suggests that these cells may be a critical population in promoting Th2 cytokine-associated inflammation in a variety of inflammatory or infectious settings. Collectively, these data suggest that the TSLP-basophil pathway may represent a new target in the design of therapeutic intervention strategies to promote or limit Th2 cytokine-dependent immunity and inflammation.

Macrophages, a key cellular component of inflammation, become functionally polarized in a signal- and context-specific manner. Th2 cytokines such as interleukin 4 (IL-4) polarize macrophages to a state of alternative activation that limits inflammation and promotes wound healing. Alternative activation is mediated by a transcriptional program that is influenced by epigenomic modifications, including histone acetylation. Here we report that macrophages lacking histone deacetylase 3 (HDAC3) display a polarization phenotype similar to IL-4-induced alternative activation and, furthermore, are hyperresponsive to IL-4 stimulation. Throughout the macrophage genome, HDAC3 deacetylates histone tails at regulatory regions, leading to repression of many IL-4-regulated genes characteristic of alternative activation. Following exposure to Schistosoma mansoni eggs, a model of Th2 cytokine-mediated disease that is limited by alternative activation, pulmonary inflammation was ameliorated in mice lacking HDAC3 in macrophages. Thus, HDAC3 functions in alternative activation as a brake whose release could be of benefit in the treatment of multiple inflammatory diseases.

Regulatory T cells (T(reg) cells) have a critical role in the maintenance of immunological self-tolerance. Here we show that treatment of naive human or mouse T cells with IL-35 induced a regulatory population, which we call 'iT(R)35 cells', that mediated suppression via IL-35 but not via the inhibitory cytokines IL-10 or transforming growth factor-β (TGF-β). We found that iT(R)35 cells did not express or require the transcription factor Foxp3, and were strongly suppressive and stable in vivo. T(reg) cells induced the generation of iT(R)35 cells in an IL-35- and IL-10-dependent manner in vitro and induced their generation in vivo under inflammatory conditions in intestines infected with Trichuris muris and within the tumor microenvironment (B16 melanoma and MC38 colorectal adenocarcinoma), where they contributed to the regulatory milieu. Thus, iT(R)35 cells constitute a key mediator of infectious tolerance and contribute to T(reg) cell-mediated tumor progression. Furthermore, iT(R)35 cells generated ex vivo might have therapeutic utility.

Dendritic cells can prime naive CD4+ T cells; however, here we demonstrate that dendritic cell-mediated priming was insufficient for the development of T helper type 2 cell-dependent immunity. We identify basophils as a dominant cell population that coexpressed major histocompatibility complex class II and interleukin 4 message after helminth infection. Basophilia was promoted by thymic stromal lymphopoietin, and depletion of basophils impaired immunity to helminth infection. Basophils promoted antigen-specific CD4+ T cell proliferation and interleukin 4 production in vitro, and transfer of basophils augmented the population expansion of helminth-responsive CD4+ T cells in vivo. Collectively, our studies suggest that major histocompatibility complex class II-dependent interactions between basophils and CD4+ T cells promote T helper type 2 cytokine responses and immunity to helminth infection.

Survival of parasitic helminths within a host requires immune evasion and excretory/secretory (ES) proteins may contribute to this process. Eosinophils are important effector cells in immunity of mice to the nematode Nippostrongylus brasiliensis and eosinophilic interleukin-5 transgenic (IL-5 Tg) mice are highly resistant to the earliest stages of primary infections. In contrast, Toxocara canis is largely resistant to eosinophils, with viable larvae encysted in tissues often surrounded by these and other leucocytes. The aim of this study was to investigate whether T. canis ES (TES) proteins inhibit eosinophil-dependent resistance to N. brasiliensis. Mouse serum pre-treated with TES had reduced capacity to mediate the adherence of leucocytes to N. brasiliensis infective-stage larvae (L3) and this correlated with reduced complement C3 deposition on the parasite. TES did not inhibit eosinophil survival or eotaxin-dependent eosinophil migration in vitro. Cellular inflammation and eosinophil degranulation in the skin in response to injection of L3 was also not impaired by TES. However, when TES was included with L3 in an inoculum given to IL-5 Tg mice, a greatly increased number of parasites migrated to the lung. This suggests that the early eosinophil-dependent resistance in these mice was suppressed, by mechanisms yet to be determined.

Complement may be important for immunity to infection with parasitic helminths, by promoting the recruitment of leukocytes to infected tissues and by modulating the function of cytotoxic effector leukocytes. However, the importance of complement in vivo during helminth infection is poorly understood. In this study, mice lacking classical (C1q-deficient), alternative (factor B-deficient) or all pathways of complement activation (C3-deficient) were used to assess the role of complement in immunity to the nematode Nippostrongylus brasiliensis. Double-mutant complement-deficient/IL-5 transgenic (Tg) mice were used to determine if complement is required for the strong eosinophil-dependent resistance to this parasite. Complement activation on larvae (C3 deposition), extracellular eosinophil peroxidase activity, larval aggregation and eosinophil recruitment to the skin 30 min post-injection (p.i.) of larvae were reduced in factor B-deficient mice. Inhibition of the C5a receptor with the antagonist PMX53 impaired eosinophil and neutrophil recruitment to the skin. C3 deposition on larvae was minimal by 150 min p.i. and at this time cell adherence, larval aggregation, eosinophil recruitment and degranulation were complement-independent. Factor B and C3 deficiency were associated with higher lung larval burdens in primary infections. Complement-deficient/IL-5 Tg mice were highly resistant to N. brasiliensis, suggesting that eosinophils can limit infection in a complement-independent manner. Potent secondary immunity was similarly complement-independent. In conclusion, although the alternative pathway is important for parasite recognition and leukocyte recruitment early in N. brasiliensis infections, the parasite soon becomes resistant to complement and other factors can compensate to promote eosinophil-dependent immunity.

Eosinophils are an important feature of immune responses to infections with many of the tissue-invasive helminth parasites. The cytokine IL-5 and a high-affinity double GATA-binding site within the GATA-1 promoter are critical for eosinophilopoiesis. In this study, we believe we demonstrate for the first time that defects in eosinophilopoiesis are associated with impaired resistance to Nippostrongylus brasiliensis. Primary and secondary infections were established in wildtype (WT), IL-5(-/-) and DeltadblGATA mice. Resistance to secondary infections was impaired in IL-5(-/-) and DeltadblGATA mice, with significantly more larvae able to reach the lungs 2 days p.i. Pulmonary inflammation was minimal in all strains in the first 2 days of both primary and secondary infections, suggesting that eosinophil-dependent resistance occurred before larvae reached this site. Intestinal worm burdens and/or parasite egg production in primary infections were greater in animals with defective eosinophilopoiesis. While larvae did reach the gut by day 3 of secondary infections of WT and IL-5(-/-) mice, worms were expelled by day 7, even in the complete absence of eosinophils in tissues of the small intestine. This and our previous studies indicate that N. brasiliensis are likely to be exquisitely sensitive to attack by eosinophils soon after entry into the skin. Eosinophils in the gut may make a modest contribution to resistance on first exposure to the parasite, but are not required for expulsion in either primary or secondary infections. In order to mount an effective immune response it may be vital for the host to identify and attack the parasite before it implements immune evasion strategies and migrates to other anatomical sites. These observations may be of particular significance for the development of successful vaccines against hookworms and other nematodes.

Complement activation and C3 deposition on the surface of parasitic helminths may be important for recruitment of leukocytes and for damage to the target organism via cell-mediated mechanisms. Inhibition of complement activation would therefore be advantageous to parasites, minimizing damage and enhancing migration through tissues. The aim of this study was to determine ex vivo if complement activation by, and leukocyte adherence to, the nematode Nippostrongylus brasiliensis change as the parasite matures and migrates through the murine host. Pathways of activation of complement and the mechanism of adherence of leukocytes were also defined using sera from mice genetically deficient in either C1q, factor B, C1q and factor B, C3, or C4. Substantive deposition of C3 and adherence of eosinophil-rich leukocytes were seen with infective-stage (L3) but not with lung-stage (L4) larvae. Adult intestinal worms had low to intermediate levels of both C3 and leukocyte binding. For L3 and adult worms, complement deposition was principally dependent on the alternative pathway. For lung-stage larvae, the small amount of C3 detected was dependent to similar degrees on both the lectin and alternative pathways. The classical pathway was not involved for any of the life stages of the parasite. These results suggest that in primary infections, the infective stage of N. brasiliensis is vulnerable to complement-dependent attack by leukocytes. However, within the first 24 h of infection, N. brasiliensis acquires the ability to largely avoid complement-dependent immune responses.

Methods used to quantify complement deposition and cell adherence to parasitic helminths usually involve subjective visual comparisons of immunofluorescence or time-consuming manual counting of bound cells. Such targets are relatively large and, generally, few individual organisms can be analysed. More objective and efficient radiometric assays are available, but these also have considerable disadvantages. We have developed an improved immunofluorescence-based method for quantitation of complement deposition on viable third-stage larvae of the nematode Nippostrongylus brasiliensis (L3). A similar strategy was also applied to measuring leukocyte adherence to the parasite. Fluorescein isothiocyanate (FITC)-conjugated antibodies were used to detect complement on serum-treated larvae. The adherence of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled mouse leukocytes to larvae was investigated using the same basic approach. Images of fluorescent larvae or fluorescent cells attached to larvae were generated with a Bio-Rad Molecular Imager FX and fluorescence intensity was quantified. Hundreds of larvae can be analysed simultaneously in multiple samples, and these strategies allow rapid and sensitive quantitation that is directly proportional to the amount of protein or the number of leukocytes added to cultures. These techniques may also be applicable to other large objects, organisms or biological surfaces.