IL-33-binding HpARI family homologues with divergent effects in suppressing or enhancing type 2 immune responses.

HpARI is an immunomodulatory protein secreted by the intestinal nematode Heligmosomoides polygyrus bakeri, which binds and blocks IL-33. Here, we find that the H. polygyrus bakeri genome contains three HpARI family members and that these have different effects on IL-33-dependent responses in vitro and in vivo, with HpARI1+2 suppressing and HpARI3 amplifying these responses. All HpARIs have sub-nanomolar affinity for mouse IL-33; however, HpARI3 does not block IL-33-ST2 interactions. Instead, HpARI3 stabilizes IL-33, increasing the half-life of the cytokine and amplifying responses to it in vivo. Together, these data show that H. polygyrus bakeri secretes a family of HpARI proteins with both overlapping and distinct functions, comprising a complex immunomodulatory arsenal of host-targeted proteins.

Collaborative parasitology: student partnerships in open education.

Student-faculty partnerships can drive innovation in parasitology education and outreach. We provide recommendations for building successful partnerships during the design, implementation, and impact assessment stages. We also introduce a new series of freely available educational and community outreach materials available on a platform that the parasitology community can contribute to.

Transcriptional patterns of sexual dimorphism and in host developmental programs in the model parasitic nematode Heligmosomoides bakeri.

BACKGROUND: Heligmosomoides bakeri (often mistaken for Heligmosomoides polygyrus) is a promising model for parasitic nematodes with the key advantage of being amenable to study and manipulation within a controlled laboratory environment. While draft genome sequences are available for this worm, which allow for comparative genomic analyses between nematodes, there is a notable lack of information on its gene expression. METHODS: We generated biologically replicated RNA-seq datasets from samples taken throughout the parasitic life of H. bakeri. RNA from tissue-dwelling and lumen-dwelling worms, collected under a dissection microscope, was sequenced on an Illumina platform. RESULTS: We find extensive transcriptional sexual dimorphism throughout the fourth larval and adult stages of this parasite and identify alternative splicing, glycosylation, and ubiquitination as particularly important processes for establishing and/or maintaining sex-specific gene expression in this species. We find sex-linked differences in transcription related to aging and oxidative and osmotic stress responses. We observe a starvation-like signature among transcripts whose expression is consistently upregulated in males, which may reflect a higher energy expenditure by male worms. We detect evidence of increased importance for anaerobic respiration among the adult worms, which coincides with the parasite's migration into the physiologically hypoxic environment of the intestinal lumen. Furthermore, we hypothesize that oxygen concentration may be an important driver of the worms encysting in the intestinal mucosa as larvae, which not only fully exposes the worms to their host's immune system but also shapes many of the interactions between the host and parasite. We find stage- and sex-specific variation in the expression of immunomodulatory genes and in anthelmintic targets. CONCLUSIONS: We examine how different the male and female worms are at the molecular level and describe major developmental events that occur in the worm, which extend our understanding of the interactions between this parasite and its host. In addition to generating new hypotheses for follow-up experiments into the worm's behavior, physiology, and metabolism, our datasets enable future more in-depth comparisons between nematodes to better define the utility of H. bakeri as a model for parasitic nematodes in general.

Selective control of parasitic nematodes using bioactivated nematicides.

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.

Trickle infection with Heligmosomoides polygyrus results in decreased worm burdens but increased intestinal inflammation and scarring.

Introduction: Intestinal roundworms cause chronic debilitating disease in animals, including humans. Traditional experimental models of these types of infection use a large single-dose infection. However, in natural settings, hosts are exposed to parasites on a regular basis and when mice are exposed to frequent, smaller doses of Heligmosomoides polygyrus, the parasites are cleared more quickly. Whether this more effective host response has any negative consequences for the host is not known. Results: Using a trickle model of infection, we found that worm clearance was associated with known resistance-related host responses: increased granuloma and tuft cell numbers, increased levels of granuloma IgG and decreased intestinal transit time, as well as higher serum IgE levels. However, we found that the improved worm clearance was also associated with an inflammatory phenotype in and around the granuloma, increased smooth muscle hypertrophy/hyperplasia, and elevated levels of Adamts gene expression. Discussion: To our knowledge, we are the first to identify the involvement of this protein family of matrix metalloproteinases (MMPs) in host responses to helminth infections. Our results highlight the delicate balance between parasite clearance and host tissue damage, which both contribute to host pathology. When continually exposed to parasitic worms, improved clearance comes at a cost.

Suppressive mechanisms by Heligmosomoides polygyrus-induced Tregs in C57BL/6 mice change over time and differ to that of naïve mice.

Disrupting or harnessing immune suppression is leading to new therapeutic avenues in a number of immune-related diseases. Understanding the suppressive functions of regulatory T cells (Tregs) in different environments is therefore key. Parasitic worms are strong inducers of Tregs and previous research has suggested that parasite-induced Tregs are stronger suppressors than naïve Tregs. In strains susceptible to the intestinal worm Heligmosomoides polygyrus, like C57BL/6 mice, it has been hypothesized that increased Treg suppression downregulates both Th1 and Th2 responses, leading to chronic infections and high worm burden. Here, we show that the suppressive capacity of Tregs is no different between cells from infected and/or naive animals. In vitro suppression induced by CD4+ CD25+ Tregs (Peyers' Patches or the mesenteric lymph nodes), isolated early (day 7, tissue dwelling phase) or late (day 21, luminal phase) during infection was similar to that induced by cells from naïve animals. Suppression was CTLA-4 dependent in Tregs from acute but not chronic infection or in Tregs from naïve animals. This highlights the versatility of Tregs and the importance of extensive Treg characterization prior to potential in vivo manipulation of this cell type.

Intravital imaging of eosinophils: Unwrapping the enigma.

Eosinophils are traditionally associated with allergic and parasitic inflammation. More recently, eosinophils have also been shown to have roles in diverse processes including development, intestinal health, thymic selection, and B-cell survival with the majority of these insights being derived from murine models and in vitro assays. Despite this, tools to measure the dynamic activity of eosinophils in situ have been lacking. Intravital microscopy is a powerful tool that enables direct visualization of leukocytes and their dynamic behavior in real-time in a wide range of processes in both health and disease. Until recently eosinophil researchers have not been able to take full advantage of this technology due to a lack of tools such as genetically encoded reporter mice. This mini-review examines the history of intravital microscopy with a focus on eosinophils. The development and use of eosinophil-specific Cre (EoCre) mice to create GFP and tdTomato fluorescent reporter animals is also described. Genetically encoded eosinophil reporter mice combined with intravital microscopy provide a powerful tool to add to the toolbox of technologies that will help us unravel the mysteries still surrounding this cell.

Eosinophils and Macrophages within the Th2-Induced Granuloma: Balancing Killing and Healing in a Tight Space.

Granuloma formation is a key host immune response generated to confine invading pathogens and limit extensive host damage. It consists of an accumulation of host immune cells around a pathogen. This host response has been extensively studied in the context of inflammatory diseases. However, there is much less known about Th2-type granulomas generated in response to parasitic worms. Based on in vitro data, innate immune cells within the granuloma are thought to immobilize and kill parasites but also act to repair damaged tissue. Understanding this dual function is key. The two billion people and many livestock/wild animals infected with helminths demonstrate that granulomas are not effective at clearing infection. However, the lack of high mortality highlights their importance in ensuring that parasite migration/tissue damage is restricted and wound healing is effective. In this review, we define two key cellular players (macrophages and eosinophils) and their associated molecular players involved in Th2 granuloma function. To date, the underlying mechanisms remain poorly understood, which is in part due to a lack of conclusive studies. Most have been performed in vitro rather than in vivo, using cells that have not been obtained from granulomas. Experiments using genetically modified mouse strains and/or antibody/chemical-mediated cell depletion have also generated conflicting results depending on the model. We discuss the caveats of previous studies and the new tools available that will help fill the gaps in our knowledge and allow a better understanding of the balance between immune killing and healing.

Increases in helminth-induced IL-21 protein levels disappear upon sample freezing.

IL-21 is a much studied cytokine that has been implicated in the regulation of TH1, TH2, TH17 and regulatory immune responses; its signalling is a promising therapeutic target for autoimmune, inflammatory and infectious diseases. Despite its biological importance, measuring IL-21 reliably has proved difficult. ELISAs are commonly used to measure cytokines in various biological samples. However, results obtained are only as good as the quality of the sample. Here, we show that when using fresh samples, a significant increase in IL-21 was measured in the intestinal homogenate of mice infected with the intestinal worm Heligmosomoides polygyrus. This difference disappeared when samples were frozen in either liquid nitrogen for two days or at -80 °C for three weeks, with levels in both naïve and infected animals decreasing. This was not observed for the IL-13 cytokine, where freezing had no impact on levels measured. Our study highlights the importance of sample storage to measuring biomarkers. Since modulating IL-21 signalling is such an important potential therapeutic avenue, accurately measuring the levels of this cytokine is key to assessing its role in various research models and clinical settings.

Toxoplasma gondii: One Organism, Multiple Models.

Toxoplasma gondii is an intensely studied protozoan parasite. It is also used as a model organism to research additional clinically relevant human and veterinary parasites due to ease of in vitro culture and genetic manipulation. Recently, it has been developed as a model of inflammatory bowel disease, due to their similar pathologies. However, researchers vary widely in how they use T. gondii, which makes study comparisons and interpretation difficult. The aim of this review is to provide researchers with a tool to: (i) determine the appropriateness of the different T. gondii models to their research, (ii) interpret results from the wide range of study conditions, and (iii) consider new advances in technology which could improve or refine their experimental setup.

Does the arthropod microbiota impact the establishment of vector-borne diseases in mammalian hosts?

The impact of the microbiota on the immune status of its host is a source of intense research and publicity. In comparison, the effect of arthropod microbiota on vector-borne infectious diseases has received little attention. A better understanding of the vector microbiota in relation to mammalian host immune responses is vital, as it can lead to strategies that affect transmission and improve vaccine design in a field of research where few vaccines exist and effective treatment is rare. Recent demonstrations of how microbiota decrease pathogen development in arthropods, and thus alter vector permissiveness to vector-borne diseases (VBDs), have led to renewed interest. However, hypotheses on the interactions between the arthropod-derived microbiota and the mammalian hosts have yet to be addressed. Advances in DNA sequencing technology, increased yield and falling costs, mean that these studies are now feasible for many microbiologists and entomologists. Here, we distill current knowledge and put forward key questions and experimental designs to shed light on this burgeoning research topic.

Chronic HIV infection impairs nonopsonic phagocytosis of malaria parasites.

Malaria-specific immune responses are altered in HIV/malaria-coinfected individuals and are associated with higher parasite burdens and more severe clinical disease. Monocyte/macrophage phagocytosis is a major mechanism of malaria parasite clearance. We hypothesized that phagocytosis of malaria-parasitized erythrocytes is impaired in coinfected individuals and could contribute to the increased parasite burdens observed. We show that nonopsonic phagocytosis of Plasmodium falciparum parasitized erythrocytes is impaired in monocytes isolated from HIV-infected individuals. The observed defects in phagocytic capacity were rescued after 6 months of antiretroviral therapy, demonstrating the importance of HIV treatment and immune reconstitution in the context of coinfection.

HIV infection deregulates innate immunity to malaria despite combination antiretroviral therapy.

OBJECTIVE: Malaria and HIV-1 adversely interact, with HIV-positive individuals suffering higher parasite burdens and worse clinical outcomes. However, the mechanisms underlying these disease interactions are unclear. We hypothesized that HIV coinfection impairs the innate immune response to malaria, and that combination antiretroviral therapy (cART) may restore this response. Our aim was to examine the innate inflammatory response of natural killer (NK), natural killer T (NKT), and γδ T-cells isolated from the peripheral blood of HIV-infected therapy-naive donors to malaria parasites, and determine the effect of cART on these responses. METHODS: Freshly isolated peripheral blood mononuclear cells from 25 HIV-infected individuals pre-cART (month 0) and post-cART (months 3 and 6), and HIV-negative individuals at matched time-points, were cultured in the presence of Plasmodium falciparum parasitized erythrocytes. Supernatants and cells were collected to assess cytokine production and phenotypic changes. RESULTS: Compared to HIV-negative participants, NKT, NK, and γδ T-cell subsets from participants with chronic HIV infection showed marked differences, including decreased production of interferon γ (IFNγ) and tumor necrosis factor (TNF) in response to malaria parasites. IFNγ production was linked to interleukin-18 receptor (IL-18R) expression in all three cell types studied. Six months of cART provided partial cellular reconstitution but had no effect on IL-18R expression, or IFNγ and TNF production. CONCLUSION: These data suggest that HIV infection impairs the inflammatory response of innate effector cells to malaria, and that the response is not fully restored within 6 months of cART. This may contribute to higher parasite burdens and ineffective immune responses, and have implications for vaccination initiatives in coinfected individuals.

HIV infection deregulates Tim-3 expression on innate cells: combination antiretroviral therapy results in partial restoration.

BACKGROUND: The Tim-3 receptor has been implicated as a negative regulator of adaptive immune responses and has been linked to T-cell dysfunction in chronic viral infections, such as HIV. Blocking Tim-3 has been proposed as a potential therapeutic intervention in HIV infection. However, a more detailed characterization of Tim-3 expression in the presence of HIV is required before such strategies can be considered. METHODS: In this study, we investigate Tim-3 expression on innate immune cell subsets in chronic HIV-infected individuals pretherapy and posttherapy. RESULTS: We report that, pretherapy, HIV infection is associated with elevated levels of Tim-3 on resting innate lymphocytes (NK, NKT, and γδ T cells), but not resting monocytes. In the absence of HIV infection, stimulation with an inflammatory stimulus resulted in decreased Tim-3 on monocytes and increased Tim-3 on NK, NKT, and γδ T cells. However, innate cells from HIV-infected donors were significantly less responsive to stimulation. Six months of combination antiretroviral therapy (cART) restored Tim-3 levels on resting NK cells but not NKT or γδ T cells. The responses of all subsets to inflammatory stimuli were restored to some extent with cART but only reached HIV-negative control levels in monocytes and NK cells. DISCUSSION: These results demonstrate that, during HIV infection, Tim-3 expression on innate cells is dysregulated and that this dysregulation is only partially restored after 6 months of cART. Our findings suggest that Tim-3 is differentially regulated on innate immune effector cells, and have direct implications for strategies designed to block Tim-3-ligand interactions.

Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-ß pathway.

Foxp3-expressing regulatory T (T reg) cells have been implicated in parasite-driven inhibition of host immunity during chronic infection. We addressed whether parasites can directly induce T reg cells. Foxp3 expression was stimulated in naive Foxp3⁻ T cells in mice infected with the intestinal helminth Heligmosomoides polygyrus. In vitro, parasite-secreted proteins (termed H. polygyrus excretory-secretory antigen [HES]) induced de novo Foxp3 expression in fluorescence-sorted Foxp3⁻ splenocytes from Foxp3-green fluorescent protein reporter mice. HES-induced T reg cells suppressed both in vitro effector cell proliferation and in vivo allergic airway inflammation. HES ligated the transforming growth factor (TGF) ß receptor and promoted Smad2/3 phosphorylation. Foxp3 induction by HES was lost in dominant-negative TGF-ßRII cells and was abolished by the TGF-ß signaling inhibitor SB431542. This inhibitor also reduced worm burdens in H. polygyrus-infected mice. HES induced IL-17 in the presence of IL-6 but did not promote Th1 or Th2 development under any conditions. Importantly, antibody to mammalian TGF-ß did not recognize HES, whereas antisera that inhibited HES did not affect TGF-ß. Foxp3 was also induced by secreted products of Teladorsagia circumcincta, a related nematode which is widespread in ruminant animals. We have therefore identified a novel pathway through which helminth parasites may stimulate T reg cells, which is likely to be a key part of the parasite's immunological relationship with the host.

Divergent roles of IRAK4-mediated innate immune responses in two experimental models of severe malaria.

Severe malaria represents a clinical spectrum of disease. We propose that innate immune inflammatory responses to malaria play key roles in the pathogenesis and clinical outcomes of distinct severe malaria syndromes. To investigate this hypothesis, mice deficient in IRAK4, central to Toll-like receptor (TLR)-mediated signaling, were studied in two experimental models of malaria: Plasmodium berghei (PbA) and Plasmodium chabaudi (PccAS). Irak4(-/-)mice had decreased pro-inflammatory cytokine production during infection in both models. However, animals were relatively protected from PbA-associated symptoms compared with wild-type mice, whereas Irak4(-/-) animals were more susceptible to PccAS-associated disease. These results show that IRAK4-mediated innate immune inflammatory responses play critical roles in divergent clinical outcomes in murine malaria models. As such, integrated approaches, using more than one model, are required to fully understand the parasite/host interactions that characterize severe malaria, and more importantly, to fully assess the effect of adjunctive therapies targeting innate immune responses to malaria.

Disruption of Nod-like receptors alters inflammatory response to infection but does not confer protection in experimental cerebral malaria.

Research relating to host inflammatory processes during malaria infection has focused on Toll-like receptors, membrane-bound receptors implicated in innate sensing, and phagocytosis of parasitized erythrocytes by host cells. This is the first study to examine the role of Nod proteins, members of the Nod-like receptor (NLR) family of cytoplasmic proteins involved in pathogen recognition, in a murine model of cerebral malaria (Plasmodium berghei ANKA, PbA). Here, we find that nod1nod2(-/-) mice infected with PbA show no difference in survival or parasitemia compared with wild-type infected animals. However, cytokine levels, notably those associated with NLR activation including interleukin (IL)1-beta, KC, and MCP-1, and proteins linked to malaria pathogenesis, such as interferon-gamma (IFN-gamma), were decreased in the nod-1nod2(-/-) animals. We therefore demonstrate for the first time that Nod proteins are activated in response to parasites, and they play a role in regulating host inflammatory responses during malaria infection.

The origins of apicomplexan sequence innovation.

The Apicomplexa are a group of phylogenetically related parasitic protists that include Plasmodium, Cryptosporidium, and Toxoplasma. Together they are a major global burden on human health and economics. To meet this challenge, several international consortia have generated vast amounts of sequence data for many of these parasites. Here, we exploit these data to perform a systematic analysis of protein family and domain incidence across the phylum. A total of 87,736 protein sequences were collected from 15 apicomplexan species. These were compared with three protein databases, including the partial genome database, PartiGeneDB, which increases the breadth of taxonomic coverage. From these searches we constructed taxonomic profiles that reveal the extent of apicomplexan sequence diversity. Sequences without a significant match outside the phylum were denoted as apicomplexan specialized. These were collated into 9134 discrete protein families and placed in the context of the apicomplexan phylogeny, identifying the putative origin of each family. Most apicomplexan families were associated with an individual genus or species. Interestingly, many genera-specific innovations were associated with specialized host cell invasion and/or parasite survival processes. Contrastingly, those families reflecting more ancestral relationships were enriched in generalized housekeeping functions such as translation and transcription, which have diverged within the apicomplexan lineage. Protein domain searches revealed 192 domains not previously reported in apicomplexans together with a number of novel domain combinations. We highlight domains that may be important to parasite survival.

Inflammatory pathways in malaria infection: TLRs share the stage with other components of innate immunity.

Severe forms of malaria infection claim over 1 million lives annually. One aspect of severe malaria pathogenesis is an excessive or dysregulated inflammatory response to infection. With the characterization of Toll-like receptors (TLRs), which initiate inflammation upon detection of microbial products, involvement of TLRs in the host response to malaria has undergone intense investigation. While TLRs appear to mediate inflammation in malaria infection and may contribute to development of severe malaria, it is unlikely that they operate in isolation from other components of innate immunity. Here, we highlight recent findings implicating other innate immune mechanisms in the host inflammatory response to malaria, propose how they may integrate and synergize with TLR pathways, and discuss opportunities and challenges associated with anti-inflammatory adjunctive therapy for the treatment of severe malaria.