Getting around the roundworms: Identifying knowledge gaps and research priorities for the ascarids.

The ascarids are a large group of parasitic nematodes that infect a wide range of animal species. In humans, they cause neglected diseases of poverty; many animal parasites also cause zoonotic infections in people. Control measures include hygiene and anthelmintic treatments, but they are not always appropriate or effective and this creates a continuing need to search for better ways to reduce the human, welfare and economic costs of these infections. To this end, Le Studium Institute of Advanced Studies organized a two-day conference to identify major gaps in our understanding of ascarid parasites with a view to setting research priorities that would allow for improved control. The participants identified several key areas for future focus, comprising of advances in genomic analysis and the use of model organisms, especially Caenorhabditis elegans, a more thorough appreciation of the complexity of host-parasite (and parasite-parasite) communications, a search for novel anthelmintic drugs and the development of effective vaccines. The participants agreed to try and maintain informal links in the future that could form the basis for collaborative projects, and to co-operate to organize future meetings and workshops to promote ascarid research.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Background: Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. Methods: moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Results: Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Conclusion: Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

Migratory pattern of zoonotic Toxocara cati and T. canis in experimentally infected pigs.

BACKGROUND: Over a billion people are infected with Toxocara canis or T. cati, the roundworms of dogs and cats. Historically, T. canis has been considered the main species responsible for human toxocarosis, but as serodiagnosis cannot discriminate between the two species, this remains unresolved. We used pigs as a relevant large animal model for human infection to assess the migratory pattern of T. cati and T. canis. METHODS: Pigs were inoculated with T. cati or T. canis eggs or PBS (negative controls) and necropsied 14 or 31 days later. Different organs and tissues were examined for parasites and pathological changes. RESULTS: Overall, the two parasite species had a similar migration pattern reaching multiple organs and tissues, including the mesenteric lymph nodes, liver, lungs, and diaphragm. We recovered larvae of both species in the brain, suggesting that T. cati also can cause neurological toxocarosis in humans. Both species induced systemic eosinophilia and histopathological changes in the lungs, livers, and mesenteric lymph nodes. CONCLUSION: This study emphasises the importance of T. cati as a zoonotic agent and the need to develop diagnostic methods that can differentiate between sources of infection in humans.

Extracellular vesicles of the probiotic bacteria E. coli O83 activate innate immunity and prevent allergy in mice.

BACKGROUND: E. coli O83 (Colinfant Newborn) is a Gram-negative (G-) probiotic bacterium used in the clinic. When administered orally, it reduces allergic sensitisation but not allergic asthma. Intranasal administration offers a non-invasive and convenient delivery method. This route bypasses the gastrointestinal tract and provides direct access to the airways, which are the target of asthma prevention. G- bacteria such as E. coli O83 release outer membrane vesicles (OMVs) to communicate with the environment. Here we investigate whether intranasally administered E. coli O83 OMVs (EcO83-OMVs) can reduce allergic airway inflammation in mice. METHODS: EcO83-OMVs were isolated by ultracentrifugation and characterised their number, morphology (shape and size), composition (proteins and lipopolysaccharide; LPS), recognition by innate receptors (using transfected HEK293 cells) and immunomodulatory potential (in naïve splenocytes and bone marrow-derived dendritic cells; BMDCs). Their allergy-preventive effect was investigated in a mouse model of ovalbumin-induced allergic airway inflammation. RESULTS: EcO83-OMVs are spherical nanoparticles with a size of about 110 nm. They contain LPS and protein cargo. We identified a total of 1120 proteins, 136 of which were enriched in OMVs compared to parent bacteria. Proteins from the flagellum dominated. OMVs activated the pattern recognition receptors TLR2/4/5 as well as NOD1 and NOD2. EcO83-OMVs induced the production of pro- and anti-inflammatory cytokines in splenocytes and BMDCs. Intranasal administration of EcO83-OMVs inhibited airway hyperresponsiveness, and decreased airway eosinophilia, Th2 cytokine production and mucus secretion. CONCLUSIONS: We demonstrate for the first time that intranasally administered OMVs from probiotic G- bacteria have an anti-allergic effect. Our study highlights the advantages of OMVs as a safe platform for the prophylactic treatment of allergy. Video Abstract.

Bacterial contact induces polar plug disintegration to mediate whipworm egg hatching.

The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris. Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs. Egg hatching was most efficient when high densities of bacteria were bound to the poles. Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut.

Polymyxin B inhibits pro-inflammatory effects of E. coli outer membrane vesicles whilst increasing immune cell uptake and clearance.

Polymyxin B (PMB) is a peptide based antibiotic that binds the lipid A moiety of lipopolysaccharide (LPS) with a resultant bactericidal effect. The interaction of PMB with LPS presented on outer membrane vesicles (OMVs) is not fully known, however, a sacrificial role of OMVs in protecting bacterial cells by sequestering PMB has been described. Here we assess the ability of PMB to neutralize the immune-stimulatory properties of OMVs whilst modulating the uptake of OMVs in human immune cells. We show for the first time that PMB increases immune cell uptake of Escherichia coli derived OMVs whilst inhibiting TNF and IL-1ß production. Therefore, we present a potential new role for PMB in the neutralization of OMVs via LPS masking and increased immune cell uptake.

Bacterial contact induces polar plug disintegration to mediate whipworm egg hatching.

The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris . Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Although unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs, egg hatching was most efficient in the presence of bacteria that bound poles with high density such as Staphylococcus aureus . Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut.

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility.

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Assessing the impact of Ascariasis and Trichuriasis on weight gain using a porcine model.

BACKGROUND: Infections with Ascaris lumbricoides and Trichuris trichiura remain significant contributors to the global burden of neglected tropical diseases. Infection may in particular affect child development as they are more likely to be infected with T. trichiura and/or A. lumbricoides and to carry higher worm burdens than adults. Whilst the impact of heavy infections are clear, the effects of moderate infection intensities on the growth and development of children remain elusive. Field studies are confounded by a lack of knowledge of infection history, nutritional status, presence of co-infections and levels of exposure to infective eggs. Therefore, animal models are required. Given the physiological similarities between humans and pigs but also between the helminths that infect them; A. suum and T. suis, growing pigs provide an excellent model to investigate the direct effects of Ascaris spp. and Trichuris spp. on weight gain. METHODS AND RESULTS: We employed a trickle infection protocol to mimic natural co-infection to assess the effect of infection intensity, determined by worm count (A. suum) or eggs per gram of faeces (A. suum and T. suis), on weight gain in a large pig population (n = 195) with variable genetic susceptibility. Pig body weights were assessed over 14 weeks. Using a post-hoc statistical approach, we found a negative association between weight gain and T. suis infection. For A. suum, this association was not significant after adjusting for other covariates in a multivariable analysis. Estimates from generalized linear mixed effects models indicated that a 1 kg increase in weight gain was associated with 4.4% (p = 0.00217) decrease in T. suis EPG and a 2.8% (p = 0.02297) or 2.2% (p = 0.0488) decrease in A. suum EPG or burden, respectively. CONCLUSIONS: Overall this study has demonstrated a negative association between STH and weight gain in growing pigs but also that T. suis infection may be more detrimental that A. suum on growth.

Population genomics of ancient and modern Trichuris trichiura.

The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.

Antigens from the parasitic nematode Trichuris suis induce metabolic reprogramming and trained immunity to constrain inflammatory responses in macrophages.

Regulation of macrophage (Mɸ) function can maintain tissue homeostasis and control inflammation. Parasitic worms (helminths) are potent modulators of host immune and inflammatory responses. They have evolved various strategies to promote immunosuppression, including redirecting phagocytic cells toward a regulatory phenotype. Although soluble products from the whipworm Trichuris suis (TSPs) have shown significant effects on Mɸ function, the mechanisms underlying these modulatory effects are still not well understood. In this study, we find that TSPs suppressed inflammatory cytokines (TNF and IL-6) in Mɸs stimulated with a broad panel of TLR agonists, whilst inducing IL-10. Moreover, M1 markers such as MHCII, CD86, iNOS, and TNF were downregulated in TSP-treated Mɸs, without polarizing them towards an M2-like phenotype. We showed that TSPs could establish a suppressed activation state of Mɸs lasting at least for 72 h, indicating an anti-inflammatory innate training. Moreover, we found that TSPs, via repression of intracellular TNF generation, decreased its secretion rather than interfering with the release of surface-bound TNF. Metabolic analysis showed that TSPs promote oxidative phosphorylation (OXPHOS) without affecting glycolytic rate. Collectively, these findings expand our knowledge on helminth-induced immune modulation and support future investigations into the anti-inflammatory properties of TSPs for therapeutic purposes.

Molecular epidemiology of Ascaris species recovered from humans and pigs in Cameroon.

BACKGROUND: In Cameroon, considerable research has been conducted on human ascariasis, but no studies have been undertaken to determine whether pigs contribute to the persistence of the infection in children or to unravel the evolutionary relationship between human-derived and pig-derived Ascaris. METHODS: DNA was extracted from adult Ascaris worms collected from humans and pigs. Segments of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes were sequenced and analysed for 83 worms to dissect the local transmission dynamics of Ascaris in Cameroon. RESULTS: The data showed high genetic diversity and revealed demographically expanding populations in the human and pig Ascaris samples. A restricted gene flow between Ascaris lumbricoides and Ascaris suum populations correlating with the preference for humans and pigs, respectively, as hosts was evident. Phylogenetic analyses and haplotype networks split the haplotypes into two major clusters, A and B. However, support for cross-transmission between hosts and hybridization were revealed through shared haplotypes among worms from both hosts. CONCLUSIONS: This study provides useful baseline information for future studies of the genetics of Ascaris in Cameroon and suggests that effective and sustainable control of human ascariasis should target both human and pig hosts.

Helminths and COVID-19 susceptibility, disease progression, and vaccination efficacy.

Almost 2 years into the coronavirus disease 2019 (COVID-19) pandemic, it remains to be determined how helminths interact with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We discuss how helminths may alter susceptibility to infection, COVID-19 pathology, and the efficiency of vaccines by combined analysis of available COVID-19 data and previous investigations of the effect of helminths in viral infections.

Helminth products modulate innate immune recognition of nucleic acids in systemic lupus erythematosus.

AIM: Current treatment of Systemic Lupus Erythematosus (SLE) is suboptimal and causes broad immunosuppression. Therapeutic use of helminths or helminth products has been suggested for autoimmune diseases such as SLE. In the present study, we evaluated possible immunomodulating effects of adult body fluid (ABF) from Ascaris suum on peripheral blood mononuclear cells (PBMCs) from SLE patients in an ex vivo setup. METHODS: PBMCs from SLE patients and healthy controls (HC) were isolated and stimulated ex vivo with ABF and Toll-like receptor agonists or activators of the stimulator of interferon genes (STING) or mitochondrial antiviral signaling protein (MAVS) pathways. After 24 h of incubation, the cytokine profile was analyzed using ELISA and Meso Scale Discovery techniques. RESULTS: ABF suppressed production of IL-6, TNF-α, CXCL10, and IL-10 by PBMCs from SLE patients and HCs following stimulation with specific agonists. ABF also reduced IFN-у production by stimulated PBMCs from HCs. CONCLUSIONS: Our data show that ABF has an immunomodulatory effect on the production of key cytokines in the pathogenesis of SLE. These results suggest that ABF or ABF components hold potential as a novel treatment option for SLE.

Parasite worm antigens instruct macrophages to release immunoregulatory extracellular vesicles.

Emerging evidence suggests that immune cells not only communicate with each other through cytokines, chemokines, and cell surface receptors, but also by releasing small membranous structures known as extracellular vesicles (EVs). EVs carry a variety of different molecules that can be taken up by recipient cells. Parasitic worms are well known for their immunomodulatory properties, but whether they can affect immune responses by altering EV-driven communication between host immune cells remains unclear. Here we provide evidence that stimulation of bone marrow-derived macrophages (BMDMs) with soluble products of Trichuris suis (TSPs), leads to the release of EVs with anti-inflammatory properties. Specifically, we found that EVs from TSP-pulsed BMDMs, but not those from unstimulated BMDMs can suppress TNFα and IL-6 release in LPS-stimulated BMDMs and BMDCs. However, no polarization toward M1 or M2 was observed in macrophages exposed to EVs. Moreover, EVs enhanced reactive oxygen species (ROS) production in the exposed BMDMs, which was associated with a deregulated redox homeostasis as revealed by pathway analysis of transcriptomic data. Proteomic analysis identified cytochrome p450 (CYP450) as a potential source of ROS in EVs from TSP-pulsed BMDMs. Finally, pharmacological inhibition of CYP450 activity could suppress ROS production in those BMDMs. In summary, we find that TSPs can modulate immune responses not only via direct interactions but also indirectly by eliciting the release of EVs from BMDMs that exert anti-inflammatory effects on recipient cells.

Corrigendum: Augmented COlorimetric NANoplasmonic (CONAN) Method for Grading Purity and Determine Concentration of EV Microliter Volume Solutions.

[This corrects the article DOI: 10.3389/fbioe.2019.00452.].

Emerging interactions between diet, gastrointestinal helminth infection, and the gut microbiota in livestock.

Increasing evidence suggests that nutritional manipulation of the commensal gut microbiota (GM) may play a key role in maintaining animal health and production in an era of reduced antimicrobial usage. Gastrointestinal helminth infections impose a considerable burden on animal performance, and recent studies suggest that infection may substantially alter the composition and function of the GM. Here, we discuss the potential interactions between different bioactive dietary components (prebiotics, probiotics and phytonutrients) and helminth infection on the GM in livestock. A number of recent studies suggest that host diet can strongly influence the nature of the helminth-GM interaction. Nutritional manipulation of the GM may thus impact helminth infection, and conversely infection may also influence how the GM responds to dietary interventions. Moreover, a dynamic interaction exists between helminths, the GM, intestinal immune responses, and inflammation. Deciphering the mechanisms underlying the diet-GM-helminth axis will likely inform future helminth control strategies, as well as having implications for how health-promoting feed additives, such as probiotics, can play a role in sustainable animal production.

Parasite-Probiotic Interactions in the Gut: Bacillus sp. and Enterococcus faecium Regulate Type-2 Inflammatory Responses and Modify the Gut Microbiota of Pigs During Helminth Infection.

Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and fecal microbiota, notably including an enrichment of fecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.