Lessons from studying roundworm and whipworm in the mouse: common themes and unique features.

Ascaris lumbricoides, the roundworm, and Trichuris trichiura, the whipworm, are human intestinal nematode parasites; both are soil-transmitted helminths, are often placed together in an epidemiological context and both remain neglected despite high prevalence. Our understanding of parasitic disease continues to be enhanced through animal models. Despite the similarities between whipworm and roundworm, there are key differences between the two species and these have influenced the application of their respective animal models. In the case of T. trichiura, the fact that a murine equivalent, T. muris completes its life cycle in a mouse model has greatly enhanced our knowledge of whipworm biology, pathogenicity and immunology. In contrast, A. lumbricoides and its porcine equivalent, Ascaris suum, lack a rodent model in which the life cycle is completed. However, evidence continues to accumulate demonstrating that mice represent useful models of early Ascaris infection, a key stage of the life cycle. The use of mouse models for both Ascaris and Trichuris has a long history with early pioneers discovering fundamental aspects of each parasite's biology. Novel technologies and perspectives, as outlined in this special issue, demonstrate how through the prism of mouse models, we can continue to explore the similarities and differences between roundworms and whipworms.

Anti-inflammatory effects of infliximab in mice are independent of tumour necrosis factor α neutralization.

Infliximab (IFX) has been used repeatedly in mouse preclinical models with associated claims that anti-inflammatory effects are due to inhibition of mouse tumour necrosis factor (TNF)-α. However, the mechanism of action in mice remains unclear. In this study, the binding specificity of IFX for mouse TNF-α was investigated ex vivo using enzyme-linked immunosorbent assay (ELISA), flow cytometry and Western blot. Infliximab (IFX) did not bind directly to soluble or membrane-bound mouse TNF-α nor did it have any effect on TNF-α-induced nuclear factor kappa B (NF-κB) stimulation in mouse fibroblasts. The efficacy of IFX treatment was then investigated in vivo using a TNF-α-independent Trichuris muris-induced infection model of chronic colitis. Infection provoked severe transmural colonic inflammation by day 35 post-infection. Colonic pathology, macrophage phenotype and cell death were determined. As predicted from the in-vitro data, in-vivo treatment of T. muris-infected mice with IFX had no effect on clinical outcome, nor did it affect macrophage cell phenotype or number. IFX enhanced apoptosis of colonic immune cells significantly, likely to be driven by a direct effect of the humanized antibody itself. We have demonstrated that although IFX does not bind directly to TNF-α, observed anti-inflammatory effects in other mouse models may be through host cell apoptosis. We suggest that more careful consideration of xenogeneic responses should be made when utilizing IFX in preclinical models.

The retinoic acid-producing capacity of gut dendritic cells and macrophages is reduced during persistent T. muris infection.

Trichuris muris is an intestinal nematode that invades the colonic epithelium triggering a mucosal inflammation. Vitamin A and its active metabolite retinoic acid are strongly linked with the modulation of gut immune responses. Here, we describe the temporal changes in the expression of aldehyde dehydrogenase (ALDH) enzymes, responsible for converting dietary-absorbed vitamin A into the immuno-modulatory retinoic acid in lamina propria leucocytes post-infection. We show that ALDH enzymes are expressed by both colonic macrophages and dendritic cells. Further, during an on-going T. muris infection, ALDH expression is repressed from uninfected levels and only recovers to normal levels following expulsion of the parasite. These results suggest that local regulation of cellular levels of retinoic acid is an important component of infection-driven inflammation.

Retinoic acid signalling in gastrointestinal parasite infections: lessons from mouse models.

Retinoic acid or vitamin A is important for an extensive range of biological processes, including immunomodulatory functions, however, its role in gastrointestinal parasite infections is not yet clear. Despite this, parasite infected individuals are often supplemented with vitamin A, given the co-localised prevalence of parasitic infections and vitamin deficiencies. Therefore, it is important to understand the impact of this vitamin on the immune responses to gastrointestinal parasites. Here, we review data regarding the role of retinoic acid signalling in mouse models of intestinal nematode infection, with a view to understanding better the practice of giving vitamin A supplements to worm-infected people.

Regulation of colonic epithelial cell turnover by IDO contributes to the innate susceptibility of SCID mice to Trichuris muris infection.

Tryptophan catabolism via the kynurenine pathway is dependent on the enzyme Indoleamine 2,3-dioxygenase (IDO). Expression of IDO is upregulated in a number of inflammatory settings such as wounding and infection, and the resulting local tryptophan depletion may inhibit the replication of intracellular pathogens. Indo gene expression is upregulated in the gut during chronic infection with the mouse whipworm Trichuris muris. We demonstrate an increase in the rate of colonic epithelial cell turnover after inhibition of IDO in T. muris-infected SCID mice, leading to a significant expulsion of parasite burden. We identify the goblet cell as a novel source of IDO and present data revealing a new role for IDO in the regulation of epithelial cell turnover post-infectious challenge.

Accumulation of eosinophils in intestine-draining mesenteric lymph nodes occurs after Trichuris muris infection.

Eosinophils have recently been demonstrated capable of localizing to lymph nodes that drain mucosal surfaces, in particular during T helper 2 (Th2) responses. Resistance of mice to infection with the gastrointestinal nematode Trichuris muris depends critically on mounting of a Th2 response and represents a useful model system to investigate Th2 responses. Following infection of resistant BALB/c mice with T. muris, we observed accumulation of eosinophils in intestine-draining mesenteric lymph nodes (MLNs). The accumulation of MLN eosinophils was initiated during the second week of infection and peaked during worm expulsion. In contrast, we detected a comparably late and modest increase in eosinophil numbers in the MLNs of infected susceptible AKR mice. MLN eosinophils localized preferentially to the medullary region of the lymph node, displayed an activated phenotype and contributed to the interleukin-4 (IL-4) response in the MLN. Despite this, mice genetically deficient in eosinophils efficiently generated IL-4-expressing CD4(+) T cells, produced Th2 cytokines and mediated worm expulsion during primary T. muris infection. Thus, IL-4-expressing eosinophils accumulate in MLNs of T. muris-infected BALB/c mice but are dispensable for worm expulsion and generation of Th2 responses, suggesting a distinct or subtle role of MLN eosinophils in the immune response to T. muris infection.

Arginase-1-expressing macrophages are dispensable for resistance to infection with the gastrointestinal helminth Trichuris muris.

Alternatively activated macrophages (AAMs) have key roles in the immune response to a variety of gastrointestinal helminths such as Heligmosomoides bakeri and Nippostrongylus brasiliensis. In addition, AAMs have been implicated in the resolution of infection-induced pathology in Schistosoma mansoni infection. AAMs exert their activity in part via the enzyme arginase-1 (Arg1), which hydrolyses L-arginine into urea and ornithine, and can supply precursor substrate for proline and polyamine production. Trichuris muris is a worm that resides in the large intestine with resistance being characterized by a Th2 T-cell response, which drives alternatively activated macrophage production in the local environment of the infection. To investigate the role of AAMs in T. muris infection, we used independent genetic and pharmacologic models of arginase deficiency. In acute infection and Th2-dominated immunity, arginase-deficient models expelled worms normally. Macrophage-Arg1-deficient mice showed cytokine and antibody levels comparable to wild-type animals in acute and chronic infection. We also found no role for AAMs and Arg1 in infection-induced pathology in the response to T. muris in either chronic (Th1 dominated) or acute (Th2 dominated) infections. Our data demonstrate that, unlike other gastrointestinal helminths, Arg1 expression in AAMs is not essential for resistance to T. muris in effective resolution of helminth-induced inflammation.

Cytokine-mediated regulation of chronic intestinal helminth infection.

Most inbred strains of mouse infected with the intestinal nematode Trichuris muris are resistant to infection expelling the parasite before adult worms establish. However, a few susceptible strains exist that are incapable of worm expulsion and harbor chronic infections of mature adult worms. Analyses of in vitro cytokine production by cells from the draining lymph node (mesenteric lymph node) have indicated that expulsion phenotype is tightly correlated with the selective expansion of helper T cells (Th) of the Th1 or Th2 cell subset within the mesenteric lymph node, resulting in susceptibility and resistance to T. muris, respectively. We have now confirmed and extended our in vitro observations in a series of experiments involving the in vivo manipulation of host cytokine levels. Depletion of interferon (IFN)-gamma in normally susceptible mice resulted in expulsion of the parasite, representing the first evidence for a role for IFN-gamma in the establishment of chronic helminth infection. Blocking interleukin (IL)-4 function in normally resistant animals prevented the generation of a protective immune response allowing adult stages of the parasite to develop. Conversely the administration of IL-4 to a normally susceptible host facilitated expulsion and indeed enabled established adult worms to be expelled when administered late in infection. In all cases assessment of a variety of in vivo parameters indicative of a Th1- or Th2-type response (parasite-specific immunoglobulin (Ig) G2a and the parasite-specific IgG1, total IgE levels and intestinal mastocytosis, respectively) demonstrated that the in vivo modulation of a Th1- or Th2-specific cytokine allowed the reciprocal Th cell subset to expand and become dominant with dramatic consequences for worm expulsion.

Evidence that cytokine-mediated immune interactions induced by Schistosoma mansoni alter disease outcome in mice concurrently infected with Trichuris muris.

In murine models of Schistosoma mansoni infection, egg production is associated with a switch from T helper cell (Th)1- to Th2-type responses to both schistosome-specific and unrelated antigens. Polyparasitism is common in human populations within S. mansoni endemic areas. We have, therefore, examined whether coinfection with S. mansoni could affect the outcome of a second parasitic infection, through Th2 cytokine-dependent modifications to the host immune response. We find that when mice susceptible to infection with the gut nematode Trichuris muris are coinfected with S. mansoni, they acquire the capacity to resolve T. muris infection, thus demonstrating a resistant phenotype. This ability to expel T. muris is associated with the production of Th2-associated cytokines, and corresponding antibody isotypes, in response to S. mansoni egg antigens. The Th2 response shows that there is no compartmentalization between spleen and mesenteric lymph nodes, and that the expulsion of T. muris is not caused by any changes in the host intestine associated with excretion of schistosome eggs. This influence of schistosome infections may be important, not only for the outcome of infections with unrelated pathogens in endemic areas, but also for the efficacy of vaccines in such areas.

In vitro antigen presenting cell-derived IL-10 and IL-6 correlate with Trichuris muris isolate-specific survival.

Trichuris muris, the mouse whipworm, is used as a laboratory model of the human parasite T. trichiura. Three laboratory isolates of T. muris exist - the E, J and S isolates. Previous data have shown that the S isolate survives to chronicity in C57BL/6 mice unlike the E and J isolates, which are expelled. The ability of the S isolate to persist is thought to be due to it secreting unique excretory/secretory antigens, which interact with APCs such that protective T cell responses do not develop. To determine whether APCs respond differently to E/S antigens from the three isolates we cultured isolate-specific E/S with bone marrow-derived macrophages (BMMPhi) and dendritic cells (BMDCs) in vitro. Markers of co-stimulation and levels of MHC-II were analysed by FACS and cytokine levels in supernatants quantified. E/S antigens from the S isolate consistently stimulated significantly higher levels of IL-10 and IL-6 from both macrophages (F4/80(+)CD11b(+)CD11c(-)) and dendritic cells (CD11c(+)CD11b(+)F4/80(-)) compared to J and E isolate E/S. If these in vitro differences in APC-derived cytokines, particularly IL-10, are biologically significant in vivo, they may contribute to the S isolate survival, by creating a regulatory cytokine environment in which protective immune responses are less effective.

Altered antibody responses in mannose-binding lectin-A deficient mice do not affect Trichuris muris or Schistosoma mansoni infections.

Parasitic helminths possess surface glycoconjugates that are recognized by the serum collectin molecule, mannose-binding lectin (MBL). Once bound, MBL triggers the lectin pathway of complement. Mice have two MBL, MBL-A and MBL-C. We previously showed that MBL-A deficient (MBL-A(-/-)) mice have enhanced survival of Brugia malayi microfilariae and abrogated microfilariae-specific IgM responses. In this study we show that MBL-A deficiency does not alter immunity to either Trichuris muris or Schistosoma mansoni. However, anti-nematode IgM levels were significantly lower in T. muris infected MBL-A(-/-) than wild-type mice. Interestingly nematode-specific IgG1 and IgG2a levels were higher in MBL-A(-/-) mice. Although, larval schistosomes are surrounded by a complement-sensitive membranous tegument, neither adult worm development, egg output, egg granuloma size nor cellular composition was affected in MBL-A(-/-) mice. In contrast to anti-nematode IgM responses, anti-schistosome IgM (and also IgG1 and IgG2b) responses were unaltered from wild-type mice. Anti-schistosome IgG2a was elevated, while IgG3 was significantly lowered, in MBL-A(-/-) mice. These results suggest that MBL-A is not a necessary component for immunity to either T. muris or S. mansoni helminths, however, MBL-A appears to be necessary for the development of specific IgM responses to nematode antigens.

Caveolin1 interacts with the glucocorticoid receptor in the lung but is dispensable for its anti-inflammatory actions in lung inflammation and Trichuris Muris infection.

Glucocorticoids (Gcs) are widely prescribed anti-inflammatory compounds, which act through the glucocorticoid receptor (GR). Using an unbiased proteomics screen in lung tissue, we identified the membrane protein caveolin -1 (Cav1) as a direct interaction partner of the GR. In Cav1 knockout mice GR transactivates anti-inflammatory genes, including Dusp1, more than in controls. We therefore determined the role of Cav1 in modulating Gc action in two models of pulmonary inflammation. We first tested innate responses in lung. Loss of Cav1 impaired the inflammatory response to nebulized LPS, increasing cytokine/chemokine secretion from lung, but impairing neutrophil infiltration. Despite these changes to the inflammatory response, there was no Cav1 effect on anti-inflammatory capacity of Gcs. We also tested GR/Cav1 crosstalk in a model of allergic airway inflammation. Cav1 had a very mild effect on the inflammatory response, but no effect on the Gc response - with comparable immune cell infiltrate (macrophage, eosinophils, neutrophils), pathological score and PAS positive cells observed between both genotypes. Pursuing the Th2 adaptive immune response further we demonstrate that Cav1 knockout mice retained their ability to expel the intestinal nematode parasite T.muris, which requires adaptive Th2 immune response for elimination. Therefore, Cav1 regulates innate immune responses in the lung, but does not have an effect on Th2-mediated adaptive immunity in lung or gut. Although we demonstrate that Cav1 regulates GR transactivation of anti-inflammatory genes, this does not translate to an effect on suppression of inflammation in vivo.

Mechanisms of leucocyte recruitment to the inflamed large intestine: redundancy in integrin and addressin usage.

The caecal-dwelling nematode Trichuris muris provides a natural model of human whipworm infection. Resistance to T. muris is dependent on a host Th2 response, and CD4+Th2 cells migrate to the gut-associated lymphoid tissue (GALT) to elicit parasite expulsion. Thus, CD4+T cells infiltrate the caecal lamina propria during infection, along with other leucocyte subsets that are not critical for parasite expulsion, such as eosinophils. Trafficking of leucocytes to the GALT has been shown to be dependent on the alpha4beta7/MAdCAM-1 integrin-addressin interaction. However, where inflammation is present, such as during T. muris infection, redundant mechanisms of leucocyte recruitment may also occur in addition to traditional gut-homing interactions. We utilized an anti-integrin/addressin antibody treatment regime to investigate this redundancy in resistant, T. muris-infected C57BL/6 mice. Where only the alpha4beta7/MAdCAM-1 interaction was blocked, mice remained resistant to T. muris infection, making a Th2 response and both CD4+T cells and eosinophils infiltrated the site of infection. However, in the absence of available alpha4beta7 and alpha4beta1, mice became chronically infected with T. muris and mounted a more Th1-biased immune response. Interestingly, CD4+T cells, but not eosinophils, were able to infiltrate the caecum, showing different levels of redundancy between leucocyte subsets during infection.

Antigen selection for future anti-Trichuris vaccines: a comparison of cytokine and antibody responses to larval and adult antigen in a primary infection.

Trichuriasis, caused by the whipworm Trichuris trichiura, is endemic in tropical and subtropical areas, affecting approximately 1 billion people. Child anthelminthic treatment programmes are being implemented but repeated treatments are costly, may prevent the development of acquired immunity and can lead to the development of drug resistant parasites. Thus, the development of a vaccine which would lead to the acquisition of immunity at an earlier age and reduce community faecal egg output would be beneficial. Development of subunit vaccines requires the identification of protective antigens and their formulation in a suitable adjuvant. Trichuris muris is an antigenically similar laboratory model for T. trichiura. Subcutaneous vaccination with adult excretory-secretory products (ES) protects susceptible mouse strains from T. muris. Larval stages may contain novel and more relevant antigens which when incorporated in a vaccine induce worm expulsion earlier in infection than the adult worm products. This study finds negligible difference in the cellular and humoral immune response to T. muris adult and third stage larva(e) (L3) ES during a primary T. muris infection, but identifies high molecular weight proteins in both adult and L3 ES as potential vaccine candidates.

Stability in metabolic phenotypes and inferred metagenome profiles before the onset of colitis-induced inflammation.

Inflammatory bowel disease (IBD) is associated with altered microbiota composition and metabolism, but it is unclear whether these changes precede inflammation or are the result of it since current studies have mainly focused on changes after the onset of disease. We previously showed differences in mucus gut microbiota composition preceded colitis-induced inflammation and stool microbial differences only became apparent at colitis onset. In the present study, we aimed to investigate whether microbial dysbiosis was associated with differences in both predicted microbial gene content and endogenous metabolite profiles. We examined the functional potential of mucus and stool microbial communities in the mdr1a -/- mouse model of colitis and littermate controls using PICRUSt on 16S rRNA sequencing data. Our findings indicate that despite changes in microbial composition, microbial functional pathways were stable before and during the development of mucosal inflammation. LC-MS-based metabolic phenotyping (metabotyping) in urine samples confirmed that metabolite profiles in mdr1a -/- mice were remarkably unaffected by development of intestinal inflammation and there were no differences in previously published metabolic markers of IBD. Metabolic profiles did, however, discriminate the colitis-prone mdr1a -/- genotype from controls. Our results indicate resilience of the metabolic network irrespective of inflammation. Importantly as metabolites differentiated genotype, genotype-differentiating metabolites could potentially predict IBD risk.

Intestinal nematode parasites, cytokines and effector mechanisms.

Laboratory models of intestinal nematode infection have played an important role in developing our understanding of the immune mechanisms that operate against infectious agents. The type of helper T cell response that develops following infection with intestinal nematode parasites is critical to the outcome of infection. The early events that mediate polarisation of the helper T cell subsets towards either Th1 or Th2 during intestinal nematode infection are not well characterised, but it is likely that multiple factors influence the induction of a Th1 or Th2 type response, just as multiple effector mechanisms are involved in worm expulsion. Costimulatory molecules have been shown to be important in driving T helper cell development down a specific pathway as has the immediate cytokine environment during T cell activation. If helper T cells of the Th2 type gain ascendancy then a protective immune response ensues, mediated by Th2 type cytokines and the effector mechanisms they control. In contrast, if an inappropriate Th1 type response predominates the ability to expel infection is compromised. Equally important is the observation that multiple potential effector mechanisms are stimulated by nematode infection, with a unique combination operating against the parasite depending on nematode species and its life cycle stage. Despite the close association between intestinal nematode infection and the generation of eosinophilia, mastocytosis and IgE it has been difficult to consistently demonstrate a role for these effector cells/molecules in resistance to nematode parasites, although mast cells are clearly important in some cases. It therefore seems that, in general, less classical Th2 controlled effector mechanisms, which remain poorly defined, are probably important in resistance to nematode parasites. Thus, our understanding of both the induction and effector phases remains incomplete and will remain an intense area of interest in the coming years.

The effect of challenge and trickle Trichuris muris infections on the polarisation of the immune response.

In the field, determination of mechanisms of immunity to geohelminths are problematic due to the variation in infection exposure, host genetics, nutrition and co-infection. This study uses a well defined laboratory model, Trichuris muris in the mouse to study immune responses to challenge and trickle infections. The rationale is thus to study parasite acquisition under more natural antigen dose exposure. Antigen dose has previously been shown in this system to affect the outcome of infection with low antigen doses favouring type 1 responses (and susceptibility) and high antigen doses favouring type 2 responses (and resistance). A high level challenge infection could be established in a normally resistant host but only following priming of the immune response by a low level infection. Once type 2 responses were initiated it was impossible to switch an ongoing type 2 response even using IL-12 which is a potent stimulus of type 1 responses. Trickle infections resulted in no clear polarisation of the immune response. It was possible to build up the level of infection to a threshold level beyond which type 2 responses and expulsion were initiated. This threshold level was dependent upon host genetic background. Our results reveal a complex spectrum of responses and demonstrate that resistance and type 2 responses can be built up with increasing parasite exposure. The data provide compelling evidence to support a role for acquisition of acquired immunity to gastro-intestinal nematodes under complex infection patterns such as those found in the field.

Cytokine response profiles predict species-specific infection patterns in human GI nematodes.

This study investigated associations between pre-treatment cytokine expression and infection patterns, before and after de-worming, in humans exposed to two gastrointestinal nematode species. Quantitative measures of Ascaris lumbricoides and Trichuris trichiura infection (based on faecal egg counts) were estimated immediately before and 8-9 months after treatment in a Cameroonian population. Whole blood cytokine responses to parasite-derived antigens were assayed immediately pre-treatment. An overall measure of the tendency towards species-specific infection (increasing with A. lumbricoides faecal egg counts and decreasing with T. trichiura faecal egg counts) was significantly positively related to IL-10 levels in older (14-57 year) hosts. There was a significant negative influence of IL-5 on reinfection probability in T. trichiura but not A. lumbricoides. This effect coincided with reduced reinfection success in T. trichiura compared to A. lumbricoides. T(H)2 cytokine expression by younger hosts (4-13 year) was negatively associated with contemporary A. lumbricoides faecal egg counts before treatment. Following treatment, the pre-treatment T(H)2 cytokine expression data for younger hosts (now reflecting responsiveness 8-9 months in the past) were negatively associated with T. trichiura faecal egg counts. Taken together, these observations suggest a successional interaction between T(H)2-driven immune responses and species infection over time. However, any differential effects of the measured immune responses on species-specific recruitment, maturation and mortality were superimposed upon (and outweighed by) the effects of other factors favouring coinfection.

A classification of tasks for the systematic study of immune response using functional genomics data.

A full understanding of the immune system and its responses to infection by different pathogens is important for the development of anti-parasitic vaccines. A growing number of large-scale experimental techniques, such as microarrays, are being used to gain a better understanding of the immune system. To analyse the data generated by these experiments, methods such as clustering are widely used. However, individual applications of these methods tend to analyse the experimental data without taking publicly available biological and immunological knowledge into account systematically and in an unbiased manner. To make best use of the experimental investment, to benefit from existing evidence, and to support the findings in the experimental data, available biological information should be included in the analysis in a systematic manner. In this review we present a classification of tasks that shows how experimental data produced by studies of the immune system can be placed in a broader biological context. Taking into account available evidence, the classification can be used to identify different ways of analysing the experimental data systematically. We have used the classification to identify alternative ways of analysing microarray data, and illustrate its application using studies of immune responses in mice to infection with the intestinal nematode parasites Trichuris muris and Heligmosomoides polygyrus.

Have gastrointestinal nematodes outwitted the immune system?

Gastrointestinal (GI) nematodes are incredibly successful parasites. Choosing to live in an exposed extracellular niche, in confrontation with a potentially hostile environment, their persistent, chronic lifestyle is persuasive evidence in itself for their profound ability to modulate their hosts' immune response. Modulation is essential to avoid their own destruction but also subtly balanced to avoid compromising host survival. This review describes the early circumstantial evidence that gave clues to the immunomodulatory capabilities of the GI nematodes, the roles that T regulatory cells and alternatively activated macrophages play in this immunomodulation and provides examples of the types of specific parasite-derived factors that are known to modulate host immunity, potentiating parasite survival.