A genome-scale metabolic model of parasitic whipworm.

Genome-scale metabolic models are widely used to enhance our understanding of metabolic features of organisms, host-pathogen interactions and to identify therapeutics for diseases. Here we present iTMU798, the genome-scale metabolic model of the mouse whipworm Trichuris muris. The model demonstrates the metabolic features of T. muris and allows the prediction of metabolic steps essential for its survival. Specifically, that Thioredoxin Reductase (TrxR) enzyme is essential, a prediction we validate in vitro with the drug auranofin. Furthermore, our observation that the T. muris genome lacks gsr-1 encoding Glutathione Reductase (GR) but has GR activity that can be inhibited by auranofin indicates a mechanism for the reduction of glutathione by the TrxR enzyme in T. muris. In addition, iTMU798 predicts seven essential amino acids that cannot be synthesised by T. muris, a prediction we validate for the amino acid tryptophan. Overall, iTMU798 is as a powerful tool to study not only the T. muris metabolism but also other Trichuris spp. in understanding host parasite interactions and the rationale design of new intervention strategies.

High-fat diet-induced resistance to helminth infection via alternative induction of type 2 immunity.

Gastrointestinal nematode infections cause morbidity and socioeconomic loss in the most deprived communities. The shift in the context of obesity has led to spatial overlap with endemic gastrointestinal nematode regions resulting in the emergence of a novel comorbidity. Despite this, the impact of a high-fat diet (HFD) on immune-regulated protection against gastrointestinal infections remains largely unknown. We employed the murine model of nematode infection, Trichuris muris, to investigate the effect of an HFD on the immune response against chronic infection. Surprisingly, diet-induced obesity drove parasite expulsion in both single and repeated trickle low doses of T. muris eggs. Mechanistically, an HFD increased the expression of the ST2 receptor on CD4+ T cells, priming an enhanced type 2 helper T (Th2) cell cytokine production following interleukin (IL)-33 stimulation ex vivo. Despite IL-33-/- mice demonstrating that IL-33 is not critical for host protective immunity to T. muris under a conventional diet, HFD-fed T-cell deplete mice adoptively transferred with ST2-/- CD4 T cells were unable to expel a T. muris infection unlike those transferred with ST2-sufficient cells. Collectively, this study demonstrates that an HFD primes CD4+ T cells to utilize the IL-33-ST2 axis in a novel induction of type 2 immunity, providing insights into the emerging comorbidities of obesity and nematode infection.

Amino acid availability acts as a metabolic rheostat to determine the magnitude of ILC2 responses.

Group 2 innate lymphoid cells (ILC2) are functionally poised, tissue-resident lymphocytes that respond rapidly to damage and infection at mucosal barrier sites. ILC2 reside within complex microenvironments where they are subject to cues from both the diet and invading pathogens-including helminths. Emerging evidence suggests ILC2 are acutely sensitive not only to canonical activating signals but also perturbations in nutrient availability. In the context of helminth infection, we identify amino acid availability as a nutritional cue in regulating ILC2 responses. ILC2 are found to be uniquely preprimed to import amino acids via the large neutral amino acid transporters Slc7a5 and Slc7a8. Cell-intrinsic deletion of these transporters individually impaired ILC2 expansion, while concurrent loss of both transporters markedly impaired the proliferative and cytokine-producing capacity of ILC2. Mechanistically, amino acid uptake determined the magnitude of ILC2 responses in part via tuning of mTOR. These findings implicate essential amino acids as a metabolic requisite for optimal ILC2 responses within mucosal barrier tissues.

Rhythmicity of intestinal IgA responses confers oscillatory commensal microbiota mutualism.

Interactions between the mammalian host and commensal microbiota are enforced through a range of immune responses that confer metabolic benefits and promote tissue health and homeostasis. Immunoglobulin A (IgA) responses directly determine the composition of commensal species that colonize the intestinal tract but require substantial metabolic resources to fuel antibody production by tissue-resident plasma cells. Here, we demonstrate that IgA responses are subject to diurnal regulation over the course of a circadian day. Specifically, the magnitude of IgA secretion, as well as the transcriptome of intestinal IgA+ plasma cells, was found to exhibit rhythmicity. Oscillatory IgA responses were found to be entrained by time of feeding and were also found to be in part coordinated by the plasma cell-intrinsic circadian clock via deletion of the master clock gene Arntl. Moreover, reciprocal interactions between the host and microbiota dictated oscillatory dynamics among the commensal microbial community and its associated transcriptional and metabolic activity in an IgA-dependent manner. Together, our findings suggest that circadian networks comprising intestinal IgA, diet, and the microbiota converge to align circadian biology in the intestinal tract and to ensure host-microbial mutualism.

Whipworm secretions and their roles in host-parasite interactions.

Whipworm (Trichuris) is a genus of roundworms that causes gastrointestinal infections in humans and animals. Of particular interest are T. trichiura, the causative agent of human trichuriasis, a neglected tropical disease that affects 477 million people worldwide, and T. suis, the pig whipworm species, responsible for growth stunting and economic losses within the agricultural industry. The naturally occurring mouse whipworm, T. muris, has been used for decades as a model for trichuriasis, yielding knowledge on the biology of these parasites and the host response to infection. Ex vivo culture of T. muris (and to some extent, T. suis) has provided insight into the composition of the excretory/secretory (E/S) products released by worms, which include a myriad of proteins, RNAs, lipids, glycans, metabolites and extracellular vesicles. T. muris E/S has formed the basis of the search for whipworm vaccine candidates, while the immunomodulatory potential of T. suis and T. muris secretions has been investigated with the aim of improving our understanding of how these parasites modulate host immunity, as well as identifying immunomodulatory candidates with therapeutic potential in the context of inflammatory diseases. This article will review the various components found within Trichuris E/S, their potential as vaccine candidates and their immunomodulatory properties.

Anti-Trichuris mucosal responses are maintained during H. bakeri co-infection despite impaired parasite expulsion.

In endemic regions concurrent infection with multiple gastrointestinal (GI) helminth species is more common than single species infection. However, the majority of model helminth infections focus on single species infections leading to a lack of understanding of how co-infection influences anti-parasite immune responses. Here, we use a model co-infection of Trichuris muris (Tm) and Heligmosomoides bakeri (Hb) to investigate the effect of Hb on anti-Tm immune responses. We observed a complete impairment of Tm expulsion in immune competent C57BL/6 mice when co-infected with Hb. This was coupled with reduced cellularity in the colonic mesenteric lymph node (cMLN) proximal to the caecum, however, cMLN cytokine responses and caecal mucosal immune responses in co-infected mice were not significantly different from mice infected with Tm alone. Interestingly, in immune-compromised mice, we found co-infection resulted in enhanced growth and fecundity of female Tm parasites. These data suggest that during helminth-helminth co-infection, immune-independent signals between species may promote survival and growth.

Defining the early stages of intestinal colonisation by whipworms.

Whipworms are large metazoan parasites that inhabit multi-intracellular epithelial tunnels in the large intestine of their hosts, causing chronic disease in humans and other mammals. How first-stage larvae invade host epithelia and establish infection remains unclear. Here we investigate early infection events using both Trichuris muris infections of mice and murine caecaloids, the first in-vitro system for whipworm infection and organoid model for live helminths. We show that larvae degrade mucus layers to access epithelial cells. In early syncytial tunnels, larvae are completely intracellular, woven through multiple live dividing cells. Using single-cell RNA sequencing of infected mouse caecum, we reveal that progression of infection results in cell damage and an expansion of enterocytes expressing of Isg15, potentially instigating the host immune response to the whipworm and tissue repair. Our results unravel intestinal epithelium invasion by whipworms and reveal specific host-parasite interactions that allow the whipworm to establish its multi-intracellular niche.

Hatching of parasitic nematode eggs: a crucial step determining infection.

Although hatching from eggs is fundamental for nematode biology it remains poorly understood. For animal-parasitic nematodes in particular, advancement has been slow since the 1980s. Understanding such a crucial life-cycle process would greatly improve the tractability of parasitic nematodes as experimental systems, advance fundamental knowledge, and enable translational research. Here, we review the role of eggs in the nematode life cycle and the current knowledge on the hatching cascade, including the different inducing and contributing factors, and highlight specific areas of the field that remain unknown. We examine how these knowledge gaps could be addressed and discuss their potential impact and application in nematode parasite research, treatment, and control.

Sustained Post-Developmental T-Bet Expression Is Critical for the Maintenance of Type One Innate Lymphoid Cells In Vivo.

Innate lymphoid cells (ILC) play a significant role in the intestinal immune response and T-bet+ CD127+ group 1 cells (ILC1) have been linked to the pathogenesis of human inflammatory bowel disease (IBD). However, the functional importance of ILC1 in the context of an intact adaptive immune response has been controversial. In this report we demonstrate that induced depletion of T-bet using a Rosa26-Cre-ERT2 model resulted in the loss of intestinal ILC1, pointing to a post-developmental requirement of T-bet expression for these cells. In contrast, neither colonic lamina propria (cLP) ILC2 nor cLP ILC3 abundance were altered upon induced deletion of T-bet. Mechanistically, we report that STAT1 or STAT4 are not required for intestinal ILC1 development and maintenance. Mice with induced deletion of T-bet and subsequent loss of ILC1 were protected from the induction of severe colitis in vivo. Hence, this study provides support for the clinical development of an IBD treatment based on ILC1 depletion via targeting T-bet or its downstream transcriptional targets.

Correction: TGFß-activation by dendritic cells drives Th17 induction and intestinal contractility and augments the expulsion of the parasite Trichinella spiralis in mice.

[This corrects the article DOI: 10.1371/journal.ppat.1007657.].

Functional Characterization of the Oxantel-Sensitive Acetylcholine Receptor from Trichuris muris.

The human whipworm, Trichuris trichiura, is estimated to infect 289.6 million people globally. Control of human trichuriasis is a particular challenge, as most anthelmintics have a limited single-dose efficacy, with the striking exception of the narrow-spectrum anthelmintic, oxantel. We recently identified a novel ACR-16-like subunit from the pig whipworm, T. suis which gave rise to a functional acetylcholine receptor (nAChR) preferentially activated by oxantel. However, there is no ion channel described in the mouse model parasite T. muris so far. Here, we have identified the ACR-16-like and ACR-19 subunits from T. muris, and performed the functional characterization of the receptors in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. We found that the ACR-16-like subunit from T. muris formed a homomeric receptor gated by acetylcholine whereas the ACR-19 failed to create a functional channel. The subsequent pharmacological analysis of the Tmu-ACR-16-like receptor revealed that acetylcholine and oxantel were equally potent. The Tmu-ACR-16-like was more responsive to the toxic agonist epibatidine, but insensitive to pyrantel, in contrast to the Tsu-ACR-16-like receptor. These findings confirm that the ACR-16-like nAChR from Trichuris spp. is a preferential drug target for oxantel, and highlights the pharmacological difference between Trichuris species.

Trichuris muris and comorbidities - within a mouse model context.

Trichuris muris is a mouse intestinal parasitic nematode that inhabits the large intestine of its host and induces a strong immune response. The effects of this strong anti-parasite response can be found locally within the intestinal niche and also systemically, having effects on multiple organs. Additionally, the anti-parasite response can have multiple effects on infectious organisms and on microbiota that the host is harbouring. It has been shown that Th1 responses induced by T. muris can affect progression of bowel inflammation, cause colitic-like intestinal inflammation, reduce barrier function and intestinal mucosal responses. In the brain, T. muris can exacerbate stroke outcome and other neurological conditions. In the lung, T. muris can suppress airway inflammation and alter immune responses to other parasites. Additionally, T. muris induced responses can inhibit anti-tumour immunity. Although this parasite maintains a localized niche in the large intestine, its effects can be far-reaching and substantially impact other infections through modulation of bystander immune responses.

The interplay between Trichuris and the microbiota.

Parasitic worms are amongst the most common pathogens to infect humans and have a long-established history of inflicting disease in their hosts. There is a large body of evidence that states intestine-dwelling helminths ensure their survival by influencing the host immune response against them. In recent years, it has become apparent that the large and diverse microbial communities that exist in the gastrointestinal (GI) tract of the host and within the parasite itself have a pivotal role in worm survival and persistence. Using a variety of mouse models (including laboratory, germ-free and rewilded mice), there have been new insights into how bacteria and worms interact with each other; this includes the discovery that Trichuris is unable to hatch and/or infect their host in the absence of bacteria, and that these worms contain a Trichuris-specific gut microbiota. These interactions are determined in part by the capacity of the host, gut microbiota and worms to communicate via metabolites such as butyrate, which are microbially derived and have known immunoregulatory properties. By exploring the contribution of gut bacteria to worm infections and the intricate relationship that exists between them, an exciting and emerging field in whipworm parasitology is established.

Immunoregulatory molecules secreted by Trichuris muris.

Trichuris, whipworm nematode infections are prevalent in humans, domestic livestock and mammals. All share an epithelial dwelling niche and similar life cycle with the chronic infections that follow implying that immune evasion mechanisms are operating. Nematode excretory secretory (ES) products have been shown to be a rich source of immunomodulatory molecules for many species. The Trichuris muris model is a natural parasite of mice and has been used extensively to study host­parasite interactions and provides a tractable platform for investigation of the immunoregulatory capacity of whipworm ES. The present review details progress in identification of the composition of T. muris ES, immunomodulatory components and their potential mechanisms of action. The adult T. muris secretome is dominated by one protein with modulatory capacity although remains to be completely characterized. In addition, the secretome contains multiple other proteins and small molecules that have immunomodulatory potential, certainly by comparison to other Trichuris species. Moreover, T. muris-derived exosomes/exosome-like vesicles contain both protein and multiple miRNAs providing an alternate delivery process for molecules with the potential to modulate host immunity.

Intestinal helminth co-infection is an unrecognised risk factor for increased pneumococcal carriage density and invasive disease.

Infection with Streptococcus pneumoniae is the leading cause of death in children and burden of disease is greatest where helminth infections are also common. We investigated the impact of intestinal helminth co-infection on pneumococcal carriage; a risk factor for invasive disease. We used a mouse co-infection model and clinical data to assess the impact of co-infection on carriage density. Co-infection in mice was associated with increased pneumococcal carriage density and dissemination into lungs. Helminth-infected children also exhibited increased carriage density as compared to uninfected children. Anthelmintic treatment may be a cost-effective method of reducing pneumococcal disease burden in lower-income countries.

Regulatory RNAs: A Universal Language for Inter-Domain Communication.

In eukaryotes, microRNAs (miRNAs) have roles in development, homeostasis, disease and the immune response. Recent work has shown that plant and mammalian miRNAs also mediate cross-kingdom and cross-domain communications. However, these studies remain controversial and are lacking critical mechanistic explanations. Bacteria do not produce miRNAs themselves, and therefore it is unclear how these eukaryotic RNA molecules could function in the bacterial recipient. In this review, we compare and contrast the biogenesis and functions of regulatory RNAs in eukaryotes and bacteria. As a result, we discovered several conserved features and homologous components in these distinct pathways. These findings enabled us to propose novel mechanisms to explain how eukaryotic miRNAs could function in bacteria. Further understanding in this area is necessary to validate the findings of existing studies and could facilitate the use of miRNAs as novel tools for the directed remodelling of the human microbiota.

Contrasting impact of rural, versus urban, living on glucose metabolism and blood pressure in Uganda.

Background: The burden of cardiometabolic diseases, including cardiovascular diseases and diabetes, is increasing in sub-Saharan Africa and this has been linked to urbanisation. Helminths, through their immunomodulatory properties, may protect against these disorders. We hypothesised that the rural environment protects against cardiometabolic diseases and that helminths may influence rural-urban disparity of cardiometabolic disease risk. Methods: We compared metabolic parameters of individuals aged ≥10 years living in rural, high-helminth-transmission and urban, lower-helminth-transmission settings in Uganda. Cross-sectional surveys were conducted in rural Lake Victoria island communities and in urban sub-wards in Entebbe municipality. Helminth infection and outcomes, including insulin resistance (computed using the homeostatic model assessment of insulin resistance [HOMA-IR]), fasting blood glucose, fasting blood lipids, blood pressure, body mass index (BMI), waist and hip circumference, were assessed. Results: We analysed 1,898 rural and 930 urban participants. Adjusting for BMI, exercise, smoking, alcohol intake, age and sex, urban residents had lower mean fasting glucose (adjusted mean difference [95%CI] -0.13 [-0.24, -0.01] p=0.04) and HOMA-IR (-0.13 [-0.25, -0.01] p=0.04) but higher blood pressure (systolic, 4.64 [3.23, 6.06] p<0.001; diastolic, 1.89 [0.81, 2.97] p=0.001). Current helminth infection did not explain the observed differences. Conclusions: In low-income countries, rural living may protect against hypertension but impair glucose metabolism.

IL-17A both initiates, via IFNγ suppression, and limits the pulmonary type-2 immune response to nematode infection.

Nippostrongylus brasiliensis is a well-defined model of type-2 immunity but the early lung-migrating phase is dominated by innate IL-17A production. In this study, we confirm previous observations that Il17a-KO mice infected with N. brasiliensis exhibit an impaired type-2 immune response. Transcriptional profiling of the lung on day 2 of N. brasiliensis infection revealed an increased Ifng signature in Il17a-KO mice confirmed by enhanced IFNγ protein production in lung lymphocyte populations. Depletion of early IFNγ rescued type-2 immune responses in the Il17a-KO mice demonstrating that IL-17A-mediated suppression of IFNγ promotes type-2 immunity. Notably, later in infection, once the type-2 response was established, IL-17A limited the magnitude of the type-2 response. IL-17A regulation of type-2 immunity was lung-specific and infection with Trichuris muris revealed that IL-17A promotes a type-2 immune response in the lung even when infection is restricted to the intestine. Together our data reveal IL-17A as a major regulator of pulmonary type-2 immunity such that IL-17A supports early development of a protective type-2 response by suppression of IFNγ but subsequently limits excessive type-2 responses. A failure of this feedback loop may contribute to conditions such as severe asthma, characterised by combined elevation of IL-17 and type-2 cytokines.

Immunity to Soil-Transmitted Helminths: Evidence From the Field and Laboratory Models.

Infection with soil-transmitted helminths (STH) remains a major burden on global health and agriculture. Our understanding of the immunological mechanisms that govern whether an individual is resistant or susceptible to infection is derived primarily from model infections in rodents. Typically, experimental infections employ an artificially high, single bolus of parasites that leads to rapid expulsion of the primary infection and robust immunity to subsequent challenges. However, immunity in natura is generated slowly, and is only partially effective, with individuals in endemic areas retaining low-level infections throughout their lives. Therefore, there is a gap between traditional model STH systems and observations in the field. Here, we review the immune response to traditional model STH infections in the laboratory. We compare these data to studies of natural infection in humans and rodents in endemic areas, highlighting crucial differences between experimental and natural infection. We then detail the literature to date on the use of "trickle" infections to experimentally model the kinetics of natural infection.

Development of caecaloids to study host-pathogen interactions: new insights into immunoregulatory functions of Trichuris muris extracellular vesicles in the caecum.

The caecum, an intestinal appendage in the junction of the small and large intestines, displays a unique epithelium that serves as an exclusive niche for a range of pathogens including whipworms (Trichuris spp.). While protocols to grow organoids from small intestine (enteroids) and colon (colonoids) exist, the conditions to culture organoids from the caecum have yet to be described. Here, we report methods to grow, differentiate and characterise mouse adult stem cell-derived caecal organoids, termed caecaloids. We compare the cellular composition of caecaloids with that of enteroids, identifying differences in intestinal epithelial cell populations that mimic those found in the caecum and small intestine. The remarkable similarity in the intestinal epithelial cell composition and spatial conformation of caecaloids and their tissue of origin enables their use as an in vitro model to study host interactions with important caecal pathogens. Thus, exploiting this system, we investigated the responses of caecal intestinal epithelial cells to extracellular vesicles secreted/excreted by the intracellular helminth Trichuris muris. Our findings reveal novel immunoregulatory effects of whipworm extracellular vesicles on the caecal epithelium, including the downregulation of responses to nucleic acid recognition and type-I interferon signalling.