Nematode ascarosides attenuate mammalian type 2 inflammatory responses.

Mounting evidence suggests that nematode infection can protect against disorders of immune dysregulation. Administration of live parasites or their excretory/secretory (ES) products has shown therapeutic effects across a wide range of animal models for immune disorders, including asthma. Human clinical trials of live parasite ingestion for the treatment of immune disorders have produced promising results, yet concerns persist regarding the ingestion of pathogenic organisms and the immunogenicity of protein components. Despite extensive efforts to define the active components of ES products, no small molecules with immune regulatory activity have been identified from nematodes. Here we show that an evolutionarily conserved family of nematode pheromones called ascarosides strongly modulates the pulmonary immune response and reduces asthma severity in mice. Screening the inhibitory effects of ascarosides produced by animal-parasitic nematodes on the development of asthma in an ovalbumin (OVA) murine model, we found that administration of nanogram quantities of ascr#7 prevented the development of lung eosinophilia, goblet cell metaplasia, and airway hyperreactivity. Ascr#7 suppressed the production of IL-33 from lung epithelial cells and reduced the number of memory-type pathogenic Th2 cells and ILC2s in the lung, both key drivers of the pathology of asthma. Our findings suggest that the mammalian immune system recognizes ascarosides as an evolutionarily conserved molecular signature of parasitic nematodes. The identification of a nematode-produced small molecule underlying the well-documented immunomodulatory effects of ES products may enable the development of treatment strategies for allergic diseases.

Eosinophils and IL-4 Support Nematode Growth Coincident with an Innate Response to Tissue Injury.

It has become increasingly clear that the functions of eosinophils extend beyond host defense and allergy to metabolism and tissue regeneration. These influences have strong potential to be relevant in worm infections in which eosinophils are prominent and parasites rely on the host for nutrients to support growth or reproduction. The aim of this study was to investigate the mechanism underlying the observation that eosinophils promote growth of Trichinella spiralis larvae in skeletal muscle. Our results indicate that IL-4 and eosinophils are necessary for normal larval growth and that eosinophils from IL-4 competent mice are sufficient to support growth. The eosinophil-mediated effect operates in the absence of adaptive immunity. Following invasion by newborn larvae, host gene expression in skeletal muscle was compatible with a regenerative response and a shift in the source of energy in infected tissue. The presence of eosinophils suppressed local inflammation while also influencing nutrient homeostasis in muscle. Redistribution of glucose transporter 4 (GLUT4) and phosphorylation of Akt were observed in nurse cells, consistent with enhancement of glucose uptake and glycogen storage by larvae that is known to occur. The data are consistent with a mechanism in which eosinophils promote larval growth by an IL-4 dependent mechanism that limits local interferon-driven responses that otherwise alter nutrient metabolism in infected muscle. Our findings document a novel interaction between parasite and host in which worms have evolved a strategy to co-opt an innate host cell response in a way that facilitates their growth.

Eosinophils mediate protective immunity against secondary nematode infection.

Eosinophils are versatile cells that regulate innate and adaptive immunity, influence metabolism and tissue repair, and contribute to allergic lung disease. Within the context of immunity to parasitic worm infections, eosinophils are prominent yet highly varied in function. We have shown previously that when mice undergo primary infection with the parasitic nematode Trichinella spiralis, eosinophils play an important immune regulatory role that promotes larval growth and survival in skeletal muscle. In this study, we aimed to address the function of eosinophils in secondary infection with T. spiralis. By infecting eosinophil-ablated mice, we found that eosinophils are dispensable for immunity that clears adult worms or controls fecundity in secondary infection. In contrast, eosinophil ablation had a pronounced effect on secondary infection of skeletal muscle by migratory newborn larvae. Restoring eosinophils to previously infected, ablated mice caused them to limit muscle larvae burdens. Passive immunization of naive, ablated mice with sera or Ig from infected donors, together with transfer of eosinophils, served to limit the number of newborn larvae that migrated in tissue and colonized skeletal muscle. Results from these in vivo studies are consistent with earlier findings that eosinophils bind to larvae in the presence of Abs in vitro. Although our previous findings showed that eosinophils protect the parasite in primary infection, these new data show that eosinophils protect the host in secondary infection.

Eosinophil-derived IL-10 supports chronic nematode infection.

Eosinophilia is a feature of the host immune response that distinguishes parasitic worms from other pathogens, yet a discrete function for eosinophils in worm infection has been elusive. The aim of this study was to clarify the mechanism(s) underlying the striking and unexpected observation that eosinophils protect intracellular, muscle-stage Trichinella spiralis larvae against NO-mediated killing. Our findings indicate that eosinophils are specifically recruited to sites of infection at the earliest stage of muscle infection, consistent with a local response to injury. Early recruitment is essential for larval survival. By producing IL-10 at the initiation of infection, eosinophils expand IL-10(+) myeloid dendritic cells and CD4(+) IL-10(+) T lymphocytes that inhibit inducible NO synthase (iNOS) expression and protect intracellular larvae. The results document a novel immunoregulatory function of eosinophils in helminth infection, in which eosinophil-derived IL-10 drives immune responses that eventually limit local NO production. In this way, the parasite co-opts an immune response in a way that enhances its own survival.

Eosinophils preserve parasitic nematode larvae by regulating local immunity.

Eosinophils play important roles in regulation of cellular responses under conditions of homeostasis or infection. Intestinal infection with the parasitic nematode, Trichinella spiralis, induces a pronounced eosinophilia that coincides with establishment of larval stages in skeletal muscle. We have shown previously that in mouse strains in which the eosinophil lineage is ablated, large numbers of T. spiralis larvae are killed by NO, implicating the eosinophil as an immune regulator. In this report, we show that parasite death in eosinophil-ablated mice correlates with reduced recruitment of IL-4(+) T cells and enhanced recruitment of inducible NO synthase (iNOS)-producing neutrophils to infected muscle, as well as increased iNOS in local F4/80(+)CD11b(+)Ly6C(+) macrophages. Actively growing T. spiralis larvae were susceptible to killing by NO in vitro, whereas mature larvae were highly resistant. Growth of larvae was impaired in eosinophil-ablated mice, potentially extending the period of susceptibility to the effects of NO and enhancing parasite clearance. Transfer of eosinophils into eosinophil-ablated ΔdblGATA mice restored larval growth and survival. Regulation of immunity was not dependent upon eosinophil peroxidase or major basic protein 1 and did not correlate with activity of the IDO pathway. Our results suggest that eosinophils support parasite growth and survival by promoting accumulation of Th2 cells and preventing induction of iNOS in macrophages and neutrophils. These findings begin to define the cellular interactions that occur at an extraintestinal site of nematode infection in which the eosinophil functions as a pivotal regulator of immunity.

Participation of MyD88 and interleukin-33 as innate drivers of Th2 immunity to Trichinella spiralis.

Trichinella spiralis is a highly destructive parasitic nematode that invades and destroys intestinal epithelial cells, injures many different tissues during its migratory phase, and occupies and transforms myotubes during the final phase of its life cycle. We set out to investigate the role in immunity of innate receptors for potential pathogen- or danger-associated molecular patterns (PAMPs or DAMPs). Focusing on the MyD88-dependent receptors, which include Toll-like receptors (TLRs) and interleukin-1 (IL-1) family members, we found that MyD88-deficient mice expelled worms normally, while TLR2/4-deficient mice showed accelerated worm expulsion, suggesting that MyD88 was active in signaling pathways for more than one receptor during intestinal immunity. A direct role for PAMPs in TLR activation was not supported in a transactivation assay involving a panel of murine and human TLRs. Mice deficient in the IL-1 family receptor for the DAMP, IL-33 (called ST2), displayed reduced intestinal Th2 responses and impaired mast cell activation. IL-33 was constitutively expressed in intestinal epithelial cells, where it became concentrated in nuclei within 2 days of infection. Nuclear localization was an innate response to infection that occurred in intestinal regions where worms were actively migrating. Th2 responses were also compromised in the lymph nodes draining the skeletal muscles of ST2-deficient mice, and this correlated with increased larval burdens in muscle. Our results support a mechanism in which the immune system recognizes and responds to tissue injury in a way that promotes Th2 responses.

Combinatorial effects of interleukin 10 and interleukin 4 determine the progression of hepatic inflammation following murine enteric parasitic infection.

UNLABELLED: Mice lacking the immunoregulatory cytokine interleukin 10 (IL-10) develop necrotizing hepatitis after infection with Trichinella spiralis, and inflammation is dependent on the migration of intestinally activated CD4(+) T cells into the liver. Hepatic production of IL-4 is elevated in these mice, and we hypothesized that it plays a role in the development of hepatic pathology. Wild-type (WT), IL-10 knockout (KO), IL-4 KO, and IL-10/IL-4 KO mice were orally infected, and disease progression was followed by histological examination, alanine aminotransferase assays, and flow cytometric analysis of hepatocellular content. Both IL-10 KO and IL-10/IL-4 KO mice experienced hepatocellular injury, but only IL-10 KO mice advanced to a necrotic phase. Hepatic CD4(+) T cells were the major source of IL-4, and IL-10 regulated the number of intestinally-derived CD4(+)IL-4(+) cells. Sequestration of activated neutrophils in the liver required IL-4, and neutrophil depletion prevented progression to overt necrosis. Adoptive transfer of intestinal WT CD4(+) T cells inhibited neutrophil accumulation and inflammation, but their regulatory effects did not require IL-10 signaling. CONCLUSION: The absence of IL-10 led to hepatocyte injury during infection, but IL-4 was necessary for the development of neutrophil-dependent necrosis. These studies provide new insight into the combinatorial role of these cytokines and their targets in the generation and progression of hepatic inflammation.

Expulsion of secondary Trichinella spiralis infection in rats occurs independently of mucosal mast cell release of mast cell protease II.

Our aim was to elucidate the contribution of mucosal mast cells to the effector phase of a secondary immune response to Trichinella spiralis. During secondary infection, rats expel 90-99% of T. spiralis first-stage larvae from the intestine in a matter of hours. This phenomenon appears to be unique to rats and has been called rapid expulsion. Primary intestinal infection by T. spiralis induces mastocytosis, and mast cell degranulation occurs when challenged rats exhibit rapid expulsion. These observations have engendered the view that mast cells mediate rapid expulsion. In this study, we report that immunization of adult Albino Oxford rats by an infection limited to the muscle phase did not induce intestinal mastocytosis, yet such rats exhibited rapid expulsion when challenged orally. Although mastocytosis was absent, the protease unique to mucosal mast cells, rat mast cell protease II (RMCPII), was detected in sera at the time of expulsion. We further evaluated mast cell activity in neonatal rats that display rapid expulsion. Pups born to infected dams displayed rapid expulsion, and RMCPII was detected in their sera. By feeding pups parasite-specific mAbs or polyclonal Abs before challenge infection, it was possible to dissociate mast cell degranulation from parasite expulsion. These results indicate that rapid expulsion can occur in the absence of either intestinal mastocytosis or RMCPII release. Furthermore, release of RMCPII is not sufficient to cause expulsion. The data argue against a role for mast cells in the mechanism underlying the effector phase of protective immunity against T. spiralis in rats.

Eosinophil deficiency compromises parasite survival in chronic nematode infection.

Immune responses elicited by parasitic worms share many features with those of chronic allergy. Eosinophils contribute to the inflammation that occurs in both types of disease, and helminths can be damaged or killed by toxic products released by eosinophils in vitro. Such observations inform the widely held view that eosinophils protect the host against parasitic worms. The mouse is a natural host for Trichinella spiralis, a worm that establishes chronic infection in skeletal muscle. We tested the influence of eosinophils on T. spiralis infection in two mouse strains in which the eosinophil lineage is ablated. Eosinophils were prominent in infiltrates surrounding infected muscle cells of wild-type mice; however, in the absence of eosinophils T. spiralis muscle larvae died in large numbers. Parasite death correlated with enhanced IFN-gamma and decreased IL-4 production. Larval survival improved when mice were treated with inhibitors of inducible NO synthase, implicating the NO pathway in parasite clearance. Thus, the long-standing paradigm of eosinophil toxicity in nematode infection requires reevaluation, as our results suggest that eosinophils may influence the immune response in a manner that would sustain chronic infection and insure worm survival in the host population. Such a mechanism may be deployed by other parasitic worms that depend upon chronic infection for survival.

Vaccine delivery by polymeric vehicles in the mouse reproductive tract induces sustained local and systemic immunity.

Design of easily administered vaccines to protect the female reproductive tract against STIs such as HIV, HPV and HSV is a major step in improving world health standards. However, the effect of immunization routes and regimens (prime/boost) on immune response is not well-understood. Here, we present a systematic study of vaccine delivery by different routes and prime/boosting regimens to produce a robust humoral immune response in the reproductive tract. A model antigen, ovalbumin (OVA), was delivered orally or intranasally via polymer particles, and intravaginally via polymer disks to female mice. Repeated prime/boost at a single site result in high OVA-specific antibody levels in the serum for mice immunized orally (IgA) and invaginally (IgA and IgG) after 3 months. Vaginal antibody titers were the highest for mice immunized by intravaginal routes. Vaginal boosting following intranasal or oral priming did not appear to offer similar advantages to those primed intravaginally. Systemic immunization with OVA in Freund's adjuvant produced robust serum IgG levels, but little serum IgA or antibodies in the vaginal washings. All immunization schemes produced a significant level of IgG in the intestinal mucosa, with the exception of nasal priming followed by intravaginal boost with slow-releasing disks. In contrast, only immunization by nasal priming and intravaginal boost with fast-releasing disks was able to achieve significantly high intestinal IgA titers.

Divergent metabolic adaptations to intestinal parasitic nematode infection in mice susceptible or resistant to obesity.

BACKGROUND & AIMS: Diet-induced obesity results from increased ingestion of energy-dense food and sedentary lifestyle in genetically susceptible individuals. An environmental factor that may have shaped our energy homeostasis throughout evolution is parasitic nematode infection. METHODS: To test the hypothesis that a metabolically "thrifty phenotype" is advantageous during intestinal nematode infection, we compared the responses to Heligmosomoides polygyrus infection between 2 mouse strains: obesity-prone C57Bl/6J vs obesity-resistant SWR/J. Metabolic phenotyping was performed using indirect calorimetry, dual energy x-ray absorptiometry, and magnetic resonance imaging scanning. Gene expression was assessed by quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. RESULTS: Body weight was maintained in both strains during nematode infection via different mechanisms. There was no apparent change in energy expenditure between the strains; however, SWR/J mice exhibited a marked hyperphagia (calorie intake 60% higher than C57Bl/6J) to maintain body weight. The importance of hyperphagia was confirmed by severe weight loss in a group of infected SWR/J mice whose food intake was restricted to that of naïve mice. Furthermore, SWR/J mice expelled nematodes more rapidly than C57Bl/6J mice, an effect related to a T helper cell 2 immune response. CONCLUSIONS: C57Bl/6J mice are more energy efficient during parasitic nematode infection, which may explain their ability to tolerate the infection. SWR/J mice, on the other hand, require an increase in food intake to maintain energy stores during nematode infection. In addition, a strong T helper cell 2-mediated immune response that facilitates a prompt clearance of nematode infection in SWR/J mice may have evolved to conserve energy in this strain.

T-Bet independent development of IFNγ secreting natural T helper 1 cell population in the absence of Itk.

Th1, Th2, Th9 and Th17 cells are conventional CD4+ effector T cells identified as secretors of prototypical cytokines IFNγ, IL4, IL9, and IL-17A respectively. Recently, populations of natural Th17 and Th1 cells (nTh17 and nTh1) with innate-like phenotype have been identified in the thymus that are distinct from conventional Th17 and Th1 cells. The absence of the Tec family kinase Interleukin-2 inducible T cell kinase (Itk) results in T cell immunodeficiency in mice and humans. Here we show that Itk negatively regulates the development of nTh1 cells that express IFNγ in a Tbet independent manner, and whose expansion can be enhanced by IL4. Furthermore, we show that robust induction of IL4 responses during Trichinella spiralis infection enhance the presence of nTh1 cells. We conclude T cell receptor signaling via Itk controls the development of natural Th1 cells, which are expanded by the presence of IL4.

Eosinophils in Helminth Infection: Defenders and Dupes.

Eosinophilia is a central feature of the host response to helminth infection. Larval stages of parasitic worms are killed in vitro by eosinophils in the presence of specific antibodies or complement. These findings established host defense as the paradigm for eosinophil function. Recently, studies in eosinophil-ablated mouse strains have revealed an expanded repertoire of immunoregulatory functions for this cell. Other reports document crucial roles for eosinophils in tissue homeostasis and metabolism, processes that are central to the establishment and maintenance of parasitic worms in their hosts. In this review, we summarize current understanding of the significance of eosinophils at the host-parasite interface, highlighting their distinct functions during primary and secondary exposure.

A putative serine protease among the excretory-secretory glycoproteins of L1 Trichinella spiralis.

Trichinella spiralis first-stage larvae infect susceptible hosts by invading epithelial cells that line the small intestine. During this process the larva disgorges several glycoproteins that bear an unusual, highly antigenic sugar moiety, tyvelose (3,6-dideoxy arabinohexose). Monoclonal antibodies specific for tyvelose protect the intestine against infection, implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment in the intestinal epithelium. In order to investigate these glycoproteins at the molecular level, we first prepared monoclonal anti-peptide antibodies. The antibodies bind a family of glycoproteins that are present in excretory-secretory products of first-stage larvae and are delivered to epithelial cells during invasion by T. spiralis. The major species present in an affinity purified fraction of crude T. spiralis antigens were subjected to tryptic peptide digestion. De novo amino acid sequencing of the peptides using Q-TOF tandem mass spectrometry, in combination with database searches and antibody screening of an L1 cDNA library, showed that the glycoproteins are variably glycosylated homologues of the serine protease family.

IgG antibody responses to an inhaled antigen in horses with "heaves" (recurrent airway obstruction).

A controlled experimental system for the evaluation of pulmonary immune responses in horses with "heaves" (recurrent airway obstruction) has been developed. We hypothesized that the humoral immune response to an inhaled antigen in diseased horses would be different from that of healthy horses and that chronic pulmonary inflammation would bias the production of IgG isotypes in diseased horses as compared to healthy horses. Healthy and affected horses were housed in a natural challenge environment (stabled, fed dusty hay) and exposed by inhalation, to a nebulized solution of keyhole limpet hemocyanin (KLH). Sera and bronchoalveolar lavage fluids (BALFs) were collected from horses prior to and following their inhalation exposure to the antigen. Differential cell counts were performed on the cells in the BALF. An enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of IgGa, IgGb, IgG(T) and combined IgG specific for KLH in the sera and BALF. The percentages of neutrophils in the BALF of diseased horses were increased 4-6-fold over healthy horses. Combined IgG specific for KLH was significantly greater in BALF and serum from healthy compared to diseased horses. Differences in isotypes were also evident; however, only IgGb specific for KLH in the BALF was significantly increased in healthy versus diseased horses. Possible explanations for this difference include: (1) increased destruction of antigen before it could interact with lymphocytes, (2) down-regulation of IgGb production by inhibitory cytokines in diseased horses, or (3) binding of IgGb to Fc receptors on the large numbers of neutrophils in the lungs of diseased horses. In contrast to the prevailing notion that horses with heaves have exaggerated immune responses, our data suggest that diseased horses exposed to an aerosolized protein mount weaker IgG responses compared to healthy horses.

The effect of strenuous exercise on mRNA concentrations of interleukin-12, interferon-gamma and interleukin-4 in equine pulmonary and peripheral blood mononuclear cells.

The effect of strenuous exercise on the mRNA concentrations of interleukin-12p35 subunit (IL-12p35), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in equine pulmonary and peripheral blood mononuclear cells (PBMCs) was investigated. We hypothesized that strenuous exercise would suppress the expression of IL-12p35, IFN-gamma and augment the expression of IL-4. Eleven horses were randomly divided into two groups, a stall-confined control group (n=5) and an exercise-conditioned treatment group (n=6). Bronchoalveolar and PBMCs were obtained from horses in the treatment group prior to the commencement of a 9-week conditioning program and 24h after the completion of a maximum exercise test conducted in week 12. Samples were obtained simultaneously from control horses. Differential counts were performed on the bronchoalveolar lavage cells. Real-time PCR was performed on the pulmonary and PBMCs to quantitate cytokine expression using equine-specific primers and Taqman probes. Target gene expression was normalized to 18s rRNA expression. With the exception of IL-4 in the BALF cells, mRNA for the three cytokines was detected in the mononuclear cells from all horses at both sampling times. There were no significant differences in the cytokine mRNA concentrations between the two groups of horses at either of the sampling times. These findings demonstrate that strenuous treadmill exercise does not exert a deleterious effect on gene expression for IL-12p35, IFN-gamma or IL-4 when assessed in horses 24h following the intense physical activity.

Comparison of three immunoglobulin G assays for the diagnosis of failure of passive transfer of immunity in neonatal alpacas.

Measurement of serum immunoglobulin G (IgG) is used for the assessment of passive transfer of immunity in neonatal crias, with an IgG concentration <10 g/l being suggestive of failure of passive transfer (FPT). The purpose of the current study was to determine whether 3 commercially available immunologic assays yielded comparable results for IgG in alpacas. Serum samples from 91 alpacas were used and were stored frozen until batch analysis on the same day with the 3 assays. Immunoglobulin G was measured by radial immunodiffusion (RID) and 2 immunoturbidimetric (IT) assays (IT1, configured for automated chemistry analyzers; IT2, a point-of-care test). Median IgG concentrations were significantly different between the 3 assays, with the RID (median: 15 g/l) and IT1 (median: 16 g/l) assays, which used the same standard, yielding significantly higher IgG values than IT2 (median: 11 g/l). Results indicated a diagnostic discordance in 1-17% of samples at an IgG threshold of 10 g/l. Protein electrophoresis revealed that the RID and IT1 standard contained mostly albumin (>60%), whereas the IT2 standard consisted of beta and gamma globulins. The discrepant results between assays IT1 and IT2 were eliminated when the same standard was used (IT1: median 11 g/l; IT2: 10 g/l; n = 19 and 17, respectively). The IT1 assay had the highest precision, while the RID assay had the lowest. The results indicate that camelid IgG measurement is highly dependent on the assay standard and is not directly comparable between assays, potentially resulting in underdiagnosis of FPT in some crias.

Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene.

Nematode parasites of the genus Trichinella are intracellular and distinct life cycle stages invade intestinal epithelial and skeletal muscle cells. Within the genus, Trichinella spiralis and Trichinella pseudospiralis exhibit species-specific differences with respect to host-parasite complex formation and host immune modulation. Parasite excretory-secretory (ES) proteins play important roles at the host-parasite interface and are thought to underpin these differences in biology. Serine proteases are among the most abundant group of T. spiralis ES proteins and multiple isoforms of the muscle larvae-specific TspSP-1 serine protease have been identified. Recently, a similar protein (TppSP-1) in T. pseudospiralis muscle larvae was identified. Here we report the cloning and characterisation of the full-length transcript of TppSP-1 and present comparative data between TspSP-1 and TppSP-1.

IL-10 regulates movement of intestinally derived CD4+ T cells to the liver.

Diseases that affect the intestine may have hepatic manifestations, but the mechanisms involved in establishing hepatic disease secondarily remain poorly understood. We previously reported that IL-10 knockout (KO) mice developed severe necrotizing hepatitis following oral infection with Trichinella spiralis. In this study, we used this model of intestinal inflammation to further examine the role of IL-10 in regulating hepatic injury. Hepatic damage was induced by migrating newborn larvae. By delivering the parasite directly into the portal vein, we demonstrated that an ongoing intestinal immune response was necessary for the development of hepatitis. Intestinally derived CD4+ cells increased in the livers of IL-10 KO mice, and Ab-mediated blockade of MAdCAM-1 inhibited the accumulation of CD4+alpha(4)beta(7)+ cells in the liver. Moreover, adoptive transfer of intestinally primed CD4+ T cells from IL-10 KO mice caused hepatitis in infected immunodeficient animals. Conversely, transfer of wild-type donor cells reduced the severity of hepatic inflammation in IL-10 KO recipients, demonstrating regulatory activity. Our results revealed that IL-10 prevented migration of intestinal T cells to the liver and inhibited the development of hepatitis.