Immune modulation and modulators in Heligmosomoides polygyrus infection.

The intestinal nematode parasite Heligmosomoides polygyrus bakeri exerts widespread immunomodulatory effects on both the innate and adaptive immune system of the host. Infected mice adopt an immunoregulated phenotype, with abated allergic and autoimmune reactions. At the cellular level, infection is accompanied by expanded regulatory T cell populations, skewed dendritic cell and macrophage phenotypes, B cell hyperstimulation and multiple localised changes within the intestinal environment. In most mouse strains, these act to block protective Th2 immunity. The molecular basis of parasite interactions with the host immune system centres upon secreted products termed HES (H. polygyrus excretory-secretory antigen), which include a TGF-ß-like ligand that induces de novo regulatory T cells, factors that modify innate inflammatory responses, and molecules that block allergy in vivo. Proteomic and transcriptomic definition of parasite proteins, combined with biochemical identification of immunogenic molecules in resistant mice, will provide new candidate immunomodulators and vaccine antigens for future research.

Expansion of Foxp3+ regulatory T cells in mice infected with the filarial parasite Brugia malayi.

Many helminths, including Brugia malayi, are able to establish long-lived infections in immunocompetent hosts. Growing evidence suggests that the immune system's failure to eliminate parasites is at least partially due to the effects of regulatory T cells (Tregs). To test whether parasites may directly stimulate host regulatory activity, we infected mice with two key stages of B. malayi. Both mosquito-borne infective larvae and mature adults i.p. introduced were found to preferentially expand the proportion of CD25(+)Foxp3(+) cells within the CD4(+) T cell population. The induction of Foxp3 was accompanied by raised CD25, CD103, and CTLA-4 expression, and was shown to be an active process, which accompanied the introduction of live, but not dead parasites. CTLA-4 expression was also markedly higher on Foxp3(-) cells, suggesting anergized effector populations. Peritoneal lavage CD4(+)CD25(+) cells from infected mice showed similar suppressive activity in vitro to normal splenic "natural" Tregs. Both B. malayi larvae and adults were also able to induce Foxp3 expression in adoptively transferred DO11.10 T cells, demonstrating that filarial infection can influence the development of T cells specific to a third party Ag. In addition, we showed that induction was intact in IL-4R-deficient animals, in the absence of a Th2 or alternatively activated macrophage response. We conclude that filarial infections significantly skew the balance of the host immune system toward Treg expansion and activation, in a manner dependent on live parasites but independent of a concomitant Th2 response.

The secretome of the filarial parasite, Brugia malayi: proteomic profile of adult excretory-secretory products.

The secretome of a parasite in its definitive host can be considered to be its genome in trans, to the extent that secreted products encoded by the parasite fulfill their function in the host milieu. The 'extended phenotype' of the filarial parasite, Brugia malayi, is of particular interest because of the evidence that infection results in potent down-modulation of the host immune response. We collected B. malayi 'excretory-secretory' (BES) proteins from adult parasites and using a combination of shotgun LC-MS/MS and 2D gel electrophoresis, identified 80 B. malayi and two host proteins in BES, of which 31 (38%) were detectable in whole worm extract (BmA). Products which were enriched in BES relative to BmA included phosphatidylethanolamine-binding protein (PEB), leucyl aminopeptidase (LAP, homologue of ES-62 from the related filaria Acanthocheilonema viteae), N-acetylglucosaminyltransferase (GlcNAcT) and galectin-1, in addition to the previously described major surface glycoprotein, glutathione peroxidase (gp29, GPX-1) and the cytokine homologue macrophage migration inhibitory factor (MIF-1). One of the most abundant released proteins was triose phosphate isomerase (TPI), yet many other glycolytic enzymes (such as aldolase and GAPDH) were found only in the somatic extract. Among the more prominent novel products identified in BES were a set of 11 small transthyretin-like proteins, and three glutamine-rich-repeat mucin-like proteins. Notably, no evidence was found of any secreted protein corresponding to the genome of the Wolbachia endosymbiont present in B. malayi. Western blotting with anti-phosphorylcholine (PC) monoclonal antibody identified that GlcNAcT, and not the ES-62 homologue, is the major PC-bearing protein in BES, while probing with human filariasis sera showed preferential reactivity to galectin-1 and to processed forms of myotactin. Overall, this analysis demonstrates selective release of a suite of newly identified proteins not previously suspected to be involved at the host-parasite interface, and provides important new perspectives on the biology of the filarial parasite.

IL-4R signaling is required to induce IL-10 for the establishment of T(h)2 dominance.

The requirement for IL-4 to promote differentiation of naive CD4(+) T cells into T(h)2 effector cell populations was established by classical in vitro studies. More recent in vivo data, however, indicate that signaling through the IL-4R is not essential for acquisition of the T(h)2 phenotype. In order to reconcile these seemingly contradictory conclusions, we have taken advantage of the ability of the excretory/secretory antigens of the gastrointestinal nematode Nippostrongylus brasiliensis to down-regulate T(h)1 cell development and drive T(h)2 cell expansion. We show that the initial development of IL-4-producing T cells is independent of IL-4R signaling but that the subsequent expansion of IL-4-producing CD4(+) T cells in a competitive environment that also contains T(h)1 potential is positively influenced by IL-4R signaling. We find that the production of IL-10 is the key IL-4R-dependent factor required to maintain T(h)2 dominance and that in the absence of IL-4R signaling, T(h)2 expansion can only be achieved by neutralization of T(h)1 cytokines. Moreover, in the absence of IL-4R signaling, reduced IL-10 production is due to the lack in expansion of an IL-10(+) T(h)2 population, rather than a global defect in the production of IL-10 by CD4(+) T cells. Thus, the evolution of T(h)2 dominance is achieved at the expense of T(h)1 cell development, normally restrained by IL-10 in an IL-4R-dependent manner. We conclude that T(h)2 cell development in response to N. brasiliensis antigen requires both IL-4 and IL-10 to act in concert on incipient populations of both T(h)1 and T(h)2 types.

Th2 induction by Nippostrongylus secreted antigens in mice deficient in B cells, eosinophils or MHC Class I-related receptors.

The populations of immune system cells necessary for induction of the Th2 response have yet to be fully defined. Among the most consistent natural stimuli for Th2 responses are helminth parasites, such as the gastrointestinal nematode Nippostrongylus brasiliensis. Strong Th2 bias in immune responsiveness to live parasite infection can be reproduced by injection of a soluble Nippostrongylus excretory/secretory product (NES) collected from adult worm parasites in vitro. Injection of soluble NES induces a residual type-2 response (IL-13) even in IL-4-deficient mice, and drives a fully polar Th2 response in IL-5-deficient animals. A potent IL-10 response is observed irrespective of IL-4 or IL-5 gene status. While MHC Class II knockout animals fail to mount any IL-4 or IL-10 response, the Th2 bias is intact in Class I knockouts, indicating that CD8+ or NK-like T cells restricted to classical or non-classical Class I molecules do not play an essential role in Th2 induction. B cell-deficient microMT animals also show responses, which are strongly skewed to IL-4 production. Thus, Th2 induction by Nippostrongylus antigens is independent of B cells, or MHC class I presentation, and does not require a sufficiency of eosinophils in vivo.

Signal sequence analysis of expressed sequence tags from the nematode Nippostrongylus brasiliensis and the evolution of secreted proteins in parasites.

BACKGROUND: Parasitism is a highly successful mode of life and one that requires suites of gene adaptations to permit survival within a potentially hostile host. Among such adaptations is the secretion of proteins capable of modifying or manipulating the host environment. Nippostrongylus brasiliensis is a well-studied model nematode parasite of rodents, which secretes products known to modulate host immunity. RESULTS: Taking a genomic approach to characterize potential secreted products, we analyzed expressed sequence tag (EST) sequences for putative amino-terminal secretory signals. We sequenced ESTs from a cDNA library constructed by oligo-capping to select full-length cDNAs, as well as from conventional cDNA libraries. SignalP analysis was applied to predicted open reading frames, to identify potential signal peptides and anchors. Among 1,234 ESTs, 197 (~16%) contain predicted 5' signal sequences, with 176 classified as conventional signal peptides and 21 as signal anchors. ESTs cluster into 742 distinct genes, of which 135 (18%) bear predicted signal-sequence coding regions. Comparisons of clusters with homologs from Caenorhabditis elegans and more distantly related organisms reveal that the majority (65% at P < e-10) of signal peptide-bearing sequences from N. brasiliensis show no similarity to previously reported genes, and less than 10% align to conserved genes recorded outside the phylum Nematoda. Of all novel sequences identified, 32% contained predicted signal peptides, whereas this was the case for only 3.4% of conserved genes with sequence homologies beyond the Nematoda. CONCLUSIONS: These results indicate that secreted proteins may be undergoing accelerated evolution, either because of relaxed functional constraints, or in response to stronger selective pressure from host immunity.