Simon A. Babayan

Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity

In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species.

Wild immunology: WILD IMMUNOLOGY

In wild populations, individuals are regularly exposed to a wide range of pathogens. In this context, organisms must elicit and regulate effective immune responses to protect their health while avoiding immunopathology. However, most of our knowledge about the function and dynamics of immune responses comes from laboratory studies performed on inbred mice in highly controlled environments with limited exposure to infection. Natural populations, on the other hand, exhibit wide genetic and environmental diversity. We argue that now is the time for immunology to be taken into the wild. The goal of ‘wild immunology’ is to link immune phenotype with host fitness in natural environments. To achieve this requires relevant measures of immune responsiveness that are both applicable to the host–parasite interaction under study and robustly associated with measures of host and parasite fitness. Bringing immunology to nonmodel organisms and linking that knowledge host fitness, and ultimately population dynamics, will face difficult challenges, both technical (lack of reagents and annotated genomes) and statistical (variation among individuals and populations). However, the affordability of new genomic technologies will help immunologists, ecologists and evolutionary biologists work together to translate and test our current knowledge of immune mechanisms in natural systems. From this approach, ecologists will gain new insight into mechanisms relevant to host health and fitness, while immunologists will be given a measure of the real‐world health impacts of the immune factors they study. Thus, wild immunology can be the missing link between laboratory‐based immunology and human, wildlife and domesticated animal health.

CTLA-4 and CD4+CD25+ Regulatory T Cells Inhibit Protective Immunity to Filarial Parasites In Vivo

The T cell coinhibitory receptor CTLA-4 has been implicated in the down-regulation of T cell function that is a quintessential feature of chronic human filarial infections. In a laboratory model of filariasis, Litomosoides sigmodontis infection of susceptible BALB/c mice, we have previously shown that susceptibility is linked both to a CD4+CD25+ regulatory T (Treg) cell response, and to the development of hyporesponsive CD4+ T cells at the infection site, the pleural cavity. We now provide evidence that L. sigmodontis infection drives the proliferation and activation of CD4+Foxp3+ Treg cells in vivo, demonstrated by increased uptake of BrdU and increased expression of CTLA-4, Foxp3, GITR, and CD25 compared with naive controls. The greatest increases in CTLA-4 expression were, however, seen in the CD4+Foxp3− effector T cell population which contained 78% of all CD4+CTLA-4+ cells in the pleural cavity. Depletion of CD25+ cells from the pleural CD4+ T cell population did not increase their Ag-specific proliferative response in vitro, suggesting that their hyporesponsive phenotype is not directly mediated by CD4+CD25+ Treg cells. Once infection had established, killing of adult parasites could be enhanced by neutralization of CTLA-4 in vivo, but only if performed in combination with the depletion of CD25+ Treg cells. This work suggests that during filarial infection CTLA-4 coinhibition and CD4+CD25+ Treg cells form complementary components of immune regulation that inhibit protective immunity in vivo.

Filarial Parasites Develop Faster and Reproduce Earlier in Response to Host Immune Effectors That Determine Filarial Life Expectancy

During larval development, filarial nematodes adjust their lifelong reproductive strategy to the presence of anti-parasitic immune cells that determine host resistance and experimental vaccine efficacy.

B-Cell Deficiency Suppresses Vaccine-Induced Protection against Murine Filariasis but Does Not Increase the Recovery Rate for Primary Infection

ABSTRACT To establish the role of B cells and antibodies in destroying filariae, mice lacking mature B cells and therefore unable to produce antibodies were used. Litomosoides sigmodontis offers a good opportunity for this study because it is the only filarial species that completes its life cycle in mice. Its development was compared in B-cell-deficient mice (BALB/c μMT mice) and wild-type BALB/c mice in two different in vivo situations, vaccination with irradiated larvae and primary infection. In all cases, mice were challenged with subcutaneous inoculation of 40 infective larvae. Vaccine-induced protection was suppressed in B-cell-deficient mice. In these mice, eosinophils infiltrated the subcutaneous tissue normally during immunization; however, their morphological state did not change following challenge inoculation, whereas in wild-type mice the percentage of degranulated eosinophils was markedly increased. From this, it may be deduced that the eosinophil–antibody–B-cell complex is the effector mechanism of protection in vaccinated mice and that its action is fast and takes place in the subcutaneous tissue. In primary infection, the filarial survival and growth was not modified by the absence of B cells. However, no female worm had uterine microfilariae, nor did any mice develop a patent infection. In these mice, concentrations of type 1 (gamma interferon) and type 2 (interleukin-4 [IL-4], IL-5 and IL-10) cytokines in serum were lower and pleural neutrophils were more numerous. The effects of the μMT mutation therefore differ from those in B1-cell-deficient mice described on the same BALB/c background, which reveal a higher filarial recovery rate and microfilaremia. This outlines B2-cell-dependent mechanisms as favorable to the late maturation of L. sigmodontis.

Resistance and Susceptibility to Filarial Infection with Litomosoides sigmodontis Are Associated with Early Differences in Parasite Development and in Localized Immune Reactions

ABSTRACT In order to understand natural resistance to filariasis, we compared Litomosoides sigmodontis primary infection of C57BL/6 mice, which eliminate the worms before patency, and BALB/c mice, in which worms complete their development and produce microfilariae. Our analysis over the first month of infection monitoredmigration of the infective larvae from the lymph nodes to the pleural cavity, where the worms settle. Although immune responses from the mouse strains differed from the outset, the duration of lymphatic migration (4 days) and filarial recovery rates were similar, thus confirming that the proportion of larvae that develop in the host species upon infection is not influenced by host genetic variability. The majority of worms reached the adult stage in both mouse strains; however, worm growth and molting were retarded in resistant C57BL/6 mice. Surprisingly, the only immune responses detected at 60 h postinfection occurred in the susceptible mice and only upon stimulation of cells from lymph nodes draining the inoculation site with infective larva extract: massive production of interleukin-6 (IL-6) and IL-5 (the latter cytokine was previously suspected to have an effect on L. sigmodontis growth). However, between days 10 and 30 postinfection, extraordinarily high levels of type 1 and type 2 cytokines and expansion of pleural leukocyte infiltration were seen in the resistant C57BL/6 mice, explaining the destruction of worms later. Our results suggest that events early in the infection determine susceptibility or resistance to subsequent microfilarial production and a parasite strategy to use specific immune responses to its own benefit.

Extracellular Onchocerca-derived small RNAs in host nodules and blood.

BackgroundmicroRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Here we identify extracellular, parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans.MethodsSmall RNA libraries were prepared from total RNA extracted from the nodule fluid of cattle infected with Onchocerca ochengi as well as serum and plasma from humans infected with Onchocerca volvulus in Cameroon and Ghana. Parasite-derived miRNAs were identified based on the criteria that sequences unambiguously map to hairpin structures in Onchocerca genomes, do not align to the human genome and are not present in European control serum.ResultsA total of 62 mature miRNAs from 52 distinct pre-miRNA candidates were identified in nodule fluid from cattle infected with O. ochengi of which 59 are identical in the genome of the human parasite O. volvulus. Six of the extracellular miRNAs were also identified in sequencing analyses of serum and plasma from humans infected with O. volvulus. Based on sequencing analysis the abundance levels of the parasite miRNAs in serum or plasma range from 5 to 127 reads/per million total host miRNA reads identified, comparable to our previous analyses of Schistosoma mansoni and L. sigmodontis miRNAs in serum. All six of the O. volvulus miRNAs identified have orthologs in other filarial nematodes and four were identified in the serum of mice infected with L. sigmodontis.ConclusionsWe have identified parasite-derived miRNAs associated with onchocerciasis in cattle and humans. Our results confirm the conserved nature of RNA secretion by diverse nematodes. Additional species-specific small RNAs from O. volvulus may be present in serum based on the novel miRNA sequences identified in the nodule fluid. In our analyses comparison to European control serum illuminates the scope for false-positives, warranting caution in criteria that should be applied to identification of biomarkers of infection.

Phylogenomics and Analysis of Shared Genes Suggest a Single Transition to Mutualism in Wolbachia of Nematodes

Wolbachia, endosymbiotic bacteria of the order Rickettsiales, are widespread in arthropods but also present in nematodes. In arthropods, A and B supergroup Wolbachia are generally associated with distortion of host reproduction. In filarial nematodes, including some human parasites, multiple lines of experimental evidence indicate that C and D supergroup Wolbachia are essential for the survival of the host, and here the symbiotic relationship is considered mutualistic. The origin of this mutualistic endosymbiosis is of interest for both basic and applied reasons: How does a parasite become a mutualist? Could intervention in the mutualism aid in treatment of human disease? Correct rooting and high-quality resolution of Wolbachia relationships are required to resolve this question. However, because of the large genetic distance between Wolbachia and the nearest outgroups, and the limited number of genomes so far available for large-scale analyses, current phylogenies do not provide robust answers. We therefore sequenced the genome of the D supergroup Wolbachia endosymbiont of Litomosoides sigmodontis, revisited the selection of loci for phylogenomic analyses, and performed a phylogenomic analysis including available complete genomes (from isolates in supergroups A, B, C, and D). Using 90 orthologous genes with reliable phylogenetic signals, we obtained a robust phylogenetic reconstruction, including a highly supported root to the Wolbachia phylogeny between a (A + B) clade and a (C + D) clade. Although we currently lack data from several Wolbachia supergroups, notably F, our analysis supports a model wherein the putatively mutualist endosymbiotic relationship between Wolbachia and nematodes originated from a single transition event.

Future prospects and challenges of vaccines against filariasis

Filarial infections remain a major public health and socio‐economic problem across the tropics, despite considerable effort to reduce disease burden or regionally eliminate the infection with mass drug administration programmes. The sustainability of these programmes is now open to question owing to a range of issues, not least of which is emerging evidence for drug resistance. Vaccination, if developed appropriately, remains the most cost‐effective means of long‐term disease control. The rationale for the feasibility of vaccination against filarial parasites including onchocerciasis (river blindness, Onchocerca volvulus) and lymphatic filariasis (Wuchereria bancrofti or Brugia malayi) is founded on evidence from both humans and animal models for the development of protective immunity. Nonetheless, enormous challenges need to be faced in terms of overcoming parasite‐induced suppression without inducing pathology as well as the need to both recognize and tackle evolutionary and ecological obstacles to successful vaccine development. Nonetheless, new technological advances in addition to systems biology approaches offer hope that optimal immune responses can be induced that will prevent infection, disease and/or transmission.

Comparative Analysis of the Secretome from a Model Filarial Nematode (Litomosoides sigmodontis) Reveals Maximal Diversity in Gravid Female Parasites

Filarial nematodes (superfamily Filarioidea) are responsible for an annual global health burden of ∼6.3 million disability-adjusted life-years, which represents the greatest single component of morbidity attributable to helminths affecting humans. No vaccine exists for the major filarial diseases, lymphatic filariasis and onchocerciasis; in part because research on protective immunity against filariae has been constrained by the inability of the human-parasitic species to complete their lifecycles in laboratory mice. However, the rodent filaria Litomosoides sigmodontis has become a popular experimental model, as BALB/c mice are fully permissive for its development and reproduction. Here, we provide a comprehensive analysis of excretory-secretory products from L. sigmodontis across five lifecycle stages and identifications of host proteins associated with first-stage larvae (microfilariae) in the blood. Applying intensity-based quantification, we determined the abundance of 302 unique excretory-secretory proteins, of which 64.6% were present in quantifiable amounts only from gravid adult female nematodes. This lifecycle stage, together with immature microfilariae, released four proteins that have not previously been evaluated as vaccine candidates: a predicted 28.5 kDa filaria-specific protein, a zonadhesin and SCO-spondin-like protein, a vitellogenin, and a protein containing six metridin-like ShK toxin domains. Female nematodes also released two proteins derived from the obligate Wolbachia symbiont. Notably, excretory-secretory products from all parasite stages contained several uncharacterized members of the transthyretin-like protein family. Furthermore, biotin labeling revealed that redox proteins and enzymes involved in purinergic signaling were enriched on the adult nematode cuticle. Comparison of the L. sigmodontis adult secretome with that of the human-infective filarial nematode Brugia malayi (reported previously in three independent published studies) identified differences that suggest a considerable underlying diversity of potential immunomodulators. The molecules identified in L. sigmodontis excretory-secretory products show promise not only for vaccination against filarial infections, but for the amelioration of allergy and autoimmune diseases.