Lucille F. Gagliardo

Coordinated Control of Immunity to Muscle Stage Trichinella spiralis by IL-10, Regulatory T Cells, and TGF-β

We previously demonstrated that IL-10 is critical in the control of acute inflammation during development of Trichinella spiralis in the muscle. In this study, we use gene-targeted knockout mice, adoptive transfer of specific T cell populations, and in vivo Ab treatments to determine the mechanisms by which inflammation is controlled and effector T cell responses are moderated during muscle infection. We report that CD4+CD25− effector T cells, rather than CD4+CD25+ regulatory T cells, suppress inflammation by an IL-10-dependent mechanism that limits IFN-γ production and local inducible NO synthase induction. Conversely, we show that depletion of regulatory T cells during infection results in exaggerated Th2 responses. Finally, we provide evidence that, in the absence of IL-10, TGF-β participates in control of local inflammation in infected muscle and promotes parasite survival.

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-γ 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.

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.

Antibodies to tyvelose exhibit multiple modes of interference with the epithelial niche of Trichinella spiralis.

ABSTRACT Infection with the parasitic nematode Trichinella spiralis is initiated when the L1 larva invades host intestinal epithelial cells. Monoclonal antibodies specific for glycans on the larval surface and secreted glycoproteins protect the intestine against infection. Protective antibodies recognize tyvelose which caps the target glycan. In this study, we used an in vitro model of invasion to further examine the mechanism(s) by which tyvelose-specific antibodies protect epithelial cells against T. spiralis. Using cell lines that vary in susceptibility to invasion, we confirmed and clarified the results of our in vivo studies by documenting three modes of interference: exclusion of larvae from cells, encumbrance of larvae as they migrated within epithelial monolayers, and inhibition of parasite development. Excluded larvae bear cephalic caps (C. S. McVay et al., Infect. Immun. 66:1941–1945, 1998) of immune complexes that may physically block invasion or may interfere with sensory reception. Monovalent Fab fragments prepared from a tyvelose-specific antibody also excluded larvae from cells, demonstrating that antibody binding can inhibit the parasite in the absence of antigen aggregation and cap formation. In contrast, encumbered larvae caused extensive damage to the monolayer yet were not successful in establishing a niche, as reflected by their failure to molt. These results show that antibodies to tyvelose exhibit multiple modes of inhibitory activity, further implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment by T. spiralis.

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.

Molting, Ecdysis, and Reproduction of Trichinella spiralis Are Supported In Vitro by Intestinal Epithelial Cells

ABSTRACT Trichinella spiralis is an obligate parasite of animals that has an unusual intracellular life cycle. Investigation of parasitism at the cellular and molecular levels has been challenging because of a shortage of tools for in vitro cultivation of T. spiralis. We have found that T. spiralis larvae molt, ecdyse, develop to adulthood, and reproduce when they are inoculated onto cultured intestinal epithelial cells. Initially, larvae invade and migrate through cells in a monolayer (T. ManWarren, L. Gagliardo, J. Geyer, C. McVay, S. Pearce-Kelling, and J. Appleton, Infect. Immun. 65:4806-4812, 1997). During prolonged culture in Caco-2 epithelial cells, L1 larvae molted and ecdysed with efficiencies as high as 50%. Molting and ecdysis in vitro required entry of the parasite into cells; conditions that prevented entry into cells also prevented ecdysis. When larvae were inoculated at a low density and cultured for 5 to 9 days, as many as 50% of the larvae developed to adult stages. Low numbers of mature male worms with copulatory appendages were observed in these cultures. The majority of worms that survived for five or more days were unfertilized females. Low-density cultures supported development of female worms with embryos at rates of 4 to 5%. These results show that the intestinal life cycle of T. spiralis can be supported entirely by host epithelial cells. Our model should allow more detailed investigation of intracellular parasitism by T. spiralis.

Application of Monoclonal Antibodies in Functional and Comparative Investigations of Heavy-Chain Immunoglobulins in New World Camelids

ABSTRACT Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the γ chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.

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.

Participation of MyD88 and Interleukin-33 as Innate Drivers of Th2 Immunity to Trichinella spiralis

ABSTRACT 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.

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.