A sticky end for gastrointestinal helminths; the role of the mucus barrier.

Gastrointestinal (GI) nematodes are a group of successful multicellular parasites that have evolved to coexist within the intestinal niche of multiple species. It is estimated that over 10% of the world's population are chronically infected by GI nematodes, making this group of parasitic nematodes a major burden to global health. Despite the large number of affected individuals, there are few effective treatments to eradicate these infections. Research into GI nematode infections has primarily focused on defining the immunological and pathological consequences on host protection. One important but neglected aspect of host protection is mucus, and the concept that mucus is just a simple barrier is no longer tenable. In fact, mucus is a highly regulated and dynamic-secreted matrix, underpinned by a physical hydrated network of highly glycosylated mucins, which is increasingly recognized to have a key protective role against GI nematode infections. Unravelling the complex interplay between mucins, the underlying epithelium and immune cells during infection are a major challenge and are required to fully define the protective role of the mucus barrier. This review summarizes the current state of knowledge on mucins and the mucus barrier during GI nematode infections, with particular focus on murine models of infection.

Extracellular vesicles induce protective immunity against Trichuris muris.

Gastrointestinal nematodes, such as Trichuris trichiura (human whipworm), are a major source of morbidity in humans and their livestock. There is a paucity of commercially available vaccines against these parasites, and vaccine development for T. trichiura has been impeded by a lack of known host protective antigens. Experimental vaccinations with T. muris (murine whipworm) soluble Excretory/Secretory (ES) material have demonstrated that it is possible to induce protective immunity in mice; however, the potential for extracellular vesicles (EVs) as a source of antigenic material has remained relatively unexplored. Here, we demonstrate that EVs isolated from T. muris ES can induce protective immunity in mice when administered as a vaccine without adjuvant and show that the protective properties of these EVs are dependent on intact vesicles. We also identified several proteins within EV preparations that are targeted by the host antibodies following vaccination and subsequent infection with T. muris. Many of these proteins, including VWD and vitellogenin N and DUF1943-domain-containing protein, vacuolar protein sorting-associated protein 52 and TSP-1 domain-containing protein, were detected in both soluble ES and EV samples and have homologues in other parasites of medical and veterinary importance, and as such are possible protective antigens.

Changes in the mucosal barrier during acute and chronic Trichuris muris infection.

The intestinal mucosal barrier, part of the innate immune defence, is responsive to the external environment and changes in response to infection. There is disparate evidence for the epithelial and goblet cell products within the intrinsic barrier being part of a response to resolve infection. We comprehensively analysed the changes of mucosal glycoconjugates during acute and chronic infection by utilising the Trichuris muris (T. muris) model. Transcription factors, atonal homolog 1 (Math-1) and SAM pointed domain containing ETS transcription factor (Spdef) were activated during acute infection, which promoted stem cell fate towards a secretory cell phenotype. The thickness of the intermediate barrier, the carbohydrate-rich glycocalyx, composed of cell surface mucins increased with exposure to T. muris, with an increase in Muc4, Muc13 and Muc17. Overall, hypersecretion of glycoproteins into the extrinsic barrier (mediated by IL-13) via the gamma amino-butyric acid-α3 receptor (GABA-α3), was observed during acute infection. Furthermore, altered glycosylation was observed during acute and chronic infection; mucins were more highly charged during acute infection than during chronic infection. This study readdresses the changes within the mucosal barrier, in particular in the cell surface and secreted mucins during acute and chronic nematode infection.

Trichinella spiralis antigens prime mixed Th1/Th2 response but do not induce de novo generation of Foxp3+ T cells in vitro.

Many parasitic helminth infections induce Th2-type immune responses and engage the regulatory network. In this study, we specifically investigated the influence of antigens derived from different life stages of the helminth Trichinella spiralis on the polarization of naive CD4(+) T cells by dendritic cells. Results obtained from C57BL/6 mice showed that T. spiralis derived antigens have the capacity to induce bone marrow-derived dendritic cells to acquire an incompletely mature phenotype that promotes a significant proliferation of naive CD4(+) T cells and a mixed Th1/Th2 cytokine profile with the predominance of Th2 cytokines. Increased production of IL-4, IL-9, IL-10 and IL-13 accompanied increased IFN-γ. Furthermore, dendritic cells pulsed with T. spiralis antigens did not induce an increase in the population of Foxp3(+) T regulatory cells. Although other helminth antigens have demonstrated the capacity to induce de novo generation of Foxp3(+) T regulatory cells, here our in vitro studies provide no evidence that T. spiralis antigens have this capacity.

Interleukin (IL)-18 promotes the development of chronic gastrointestinal helminth infection by downregulating IL-13.

Expulsion of the gastrointestinal nematode Trichuris muris is mediated by a T helper (Th) 2 type response involving interleukin (IL)-4 and IL-13. Here we show that Th1 response-associated susceptibility involves prior activation of IL-18 and caspase-1 followed by IL-12 and interferon (IFN)-gamma in the intestine. IL-18-deficient mice are highly resistant to chronic T. muris infection and in vivo treatment of normal mice with recombinant (r)IL-18 suppresses IL-13 and IL-4 secretion but does not affect IFN-gamma. In vivo treatment of T. muris-infected IFN-gamma-deficient mice with rIL-18 demonstrated that the inhibitory effect of IL-18 on IL-13 secretion is independent of IFN-gamma. Hence, IL-18 does not function as an IFN-gamma-inducing cytokine during chronic T. muris infection but rather as a direct regulator of Th2 cytokines. These results provide the first demonstration of the critical role of IL-18 in regulating Th cell responses during gastrointestinal nematode infection.

Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper cell type 2-mediated responses.

Using a single vector targeting strategy, we have generated mice with a combined deficiency of interleukin (IL)-4 and IL-13 to clarify their roles in T helper type 2 (Th2) cell responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of the double-deficient mice with those generated by wild-type, IL-4-deficient, and IL-13-deficient mice. Using a pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that although eosinophil infiltration, immunoglobulin E, and IL-5 production are reduced in the IL-4-deficient mice and IL-13-deficient mice, they are abolished only in the combined absence of both cytokines. Furthermore, IL-4/13-deficient animals are severely impaired in their ability to expel the gastrointestinal nematode Nippostrongylus brasiliensis. Unexpectedly, N. brasiliensis-infected IL-4/13-deficient mice developed elevated IL-5 and eosinophilia, indicating that compensatory mechanisms exist for the expression of IL-5, although serum IgE remained undetectable. IL-4/13-deficient mice default to a Th1-like phenotype characterized by the expression of interferon gamma and the production of IgG2a and IgG2b. We conclude that IL-4 and IL-13 cooperate to initiate rapid Th2 cell-driven responses, and that although their functions overlap, they perform additive roles.

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.

Tumor necrosis factor alpha is a critical component of interleukin 13-mediated protective T helper cell type 2 responses during helminth infection.

In vivo manipulation of cytokine and/or cytokine receptor expression has previously shown that resistance to infection with the caecum-dwelling helminth Trichuris muris is dependent on interleukin (IL)-4 and IL-13 while susceptibility is associated with a T helper cell type 1 (Th1) cytokine response. Using gene-targeted mice deficient in tumor necrosis factor (TNF) receptor signaling and anti-TNF-alpha monoclonal antibody treatment, we have extended these studies to reveal a critical role for TNF-alpha in regulation of Th2 cytokine-mediated host protection. In vivo blockade of TNF-alpha in normally resistant mice, although not altering IL-4, IL-5, or IL-13 production in the draining lymph node, significantly delayed worm expulsion for the duration of treatment. IL-13-mediated worm expulsion in IL-4 knockout (KO) mice was also shown to be TNF-alpha dependent, and could be enhanced by administration of recombinant TNF-alpha. Furthermore, TNF receptor KO mice failed to expel T. muris, producing high levels of parasite-specific immunoglobulin G2a and the generation of a predominantly Th1 response, suggesting that the absence of TNF function from the onset of infection dramatically alters the phenotype of the response. These results provide the first demonstration of the role of TNF-alpha in regulating Th2 cytokine-mediated responses at mucosal sites, and have implications for the design of rational therapies against helminth infection and allergy.

T-bet controls intestinal mucosa immune responses via repression of type 2 innate lymphoid cell function.

Innate lymphoid cells (ILCs) play an important role in regulating immune responses at mucosal surfaces. The transcription factor T-bet is crucial for the function of ILC1s and NCR+ ILC3s and constitutive deletion of T-bet prevents the development of these subsets. Lack of T-bet in the absence of an adaptive immune system causes microbiota-dependent colitis to occur due to aberrant ILC3 responses. Thus, T-bet expression in the innate immune system has been considered to dampen pathogenic immune responses. Here, we show that T-bet plays an unexpected role in negatively regulating innate type 2 responses, in the context of an otherwise intact immune system. Selective loss of T-bet in ILCs leads to the expansion and increased activity of ILC2s, which has a functionally important impact on mucosal immunity, including enhanced protection from Trichinella spiralis infection and inflammatory colitis. Mechanistically, we show that T-bet controls the intestinal ILC pool through regulation of IL-7 receptor signalling. These data demonstrate that T-bet expression in ILCs acts as the key transcriptional checkpoint in regulating pathogenic vs. protective mucosal immune responses, which has significant implications for the understanding of the pathogenesis of inflammatory bowel diseases and intestinal infections.

A distinct role for interleukin-13 in Th2-cell-mediated immune responses.

Immune responses elicited by allergic reactions and parasitic worm infections are characterised by the induction of T helper 2 (Th2) cells. These cells secrete cytokines such as interleukin-4 (IL-4), IL-5 and IL-13, which induce the production of immunoglobulin E (IgE) and eosinophils [1,2]. Previous studies using gastrointestinal nematodes to elucidate the role of Th2-cell-mediated immune responses have demonstrated a causal relationship between T cells and worm expulsion (reviewed in [3]). Although it has been proposed that IL-4 played a central role in these responses, recent studies demonstrated that IL-4-/- mice expel the parasitic gastrointestinal nematode Nippostrongylus brasiliensis normally [4], suggesting that another T-cell mediator is required for efficient worm clearance. Using IL-13-/- mice, we have demonstrated that, unlike wild-type and IL-4-/- mice, the IL-13-/- animals failed to clear N. brasiliensis infections efficiently, despite developing a robust Th2-like cytokine response to infection. Furthermore, treatment of the IL-13-/- mice with exogenous IL-13 resulted in a reduction in the numbers of worms recovered. The IL-13-/- animals also failed to generate the goblet cell hyperplasia that normally occurs coincident with worm expulsion. This observation may link IL-13 with the production of intestinal mucus which is believed to facilitate worm expulsion. These data support a unique role for IL-13 in Th2-cell-mediated immune responses and demonstrate that IL-13 and IL-4 are not redundant.

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.

Generation of rubella virus-neutralising antibodies by vaccination with synthetic peptides.

Four short peptides from rubella virus proteins E1 and E2, predicted to contain B cell epitopes, were used to vaccinate BALB/c mice. Sera from peptide-vaccinated animals reacted with viral antigens in ELISA and three of the four induced virus-neutralising antibody (nAb) responses. Peptide PY4, in contrast to the others, induced IgG2a responses upon vaccination and stimulated spleen cells in vitro produced IFN gamma in the absence of IL-5. It was reasoned that vaccination with PY4 caused Th1 subset activation, the appropriate type of response for anti-viral immunity and hence the efficient neutralising antibody response. Presentation of peptide for vaccination proved to be as important as the sequence. Similar profiles of IgG1 and IgG2a were detected in the sera of mice vaccinated with PY4 in Freund's complete adjuvant or alum; however nAb responses were not found when alum was used.

Cytokine regulation of resistance and susceptibility to intestinal nematode infection - from host to parasite.

Intestinal nematode infection is one of the most common forms of parasite infection worldwide. Both man and domestic stock suffer considerable morbidity from these infectious agents. The majority of our current understanding of the host parasite relationship to gut dwelling nematodes comes from well-defined laboratory models. One of the most informative over recent years has been study of whipworm infection in the mouse, Trichuris muris (T. muris). Infection in inbred strains of mouse shows a spectrum of response phenotypes reflecting the variation observed under natural conditions in the wild. Resistance and worm expulsion is mediated by CD4+ T helper two cells with a dominant role for interleukin (IL)-13. The effector mechanisms responsible for worm expulsion remain undefined but new evidence suggests a role for tumour necrosis factor alpha (TNF-alpha). Susceptibility to chronic infection is mediated through a T helper 1 (Th1) response characterised through the secretion of interferon gamma (IFN-gamma). A major new role for IL-18 has been defined in induction of a Th1 response through a novel down-regulation of IL-13. Moreover, progression to chronic infection may involve the parasite itself. T. muris secretes a protein that shares epitopes with host IFN-gamma, which may interfere with host protective cytokine, mediated protection and thus, promotes its own survival.

Cytokines may give insight into mechanisms of death in sudden infant death syndrome.

There are a number of postulated causes of sudden infant death syndrome, including bacterial toxins, defects in thermoregulation and hypersensitivity. This paper formulates the hypothesis that analysis of cytokine profiles in suspected sudden infant death syndrome victims may give an insight into mechanisms of death. These cytokine profiles may also help to identify specific causes of sudden infant death syndrome or indicate that different causes act in concert in individual cases.

Colonic transcriptional profiling in resistance and susceptibility to trichuriasis: phenotyping a chronic colitis and lessons for iatrogenic helminthosis.

BACKGROUND: Helminth therapy is advocated to restore and maintain control of inflammatory responses, particularly chronic colitis. However, helminths can induce chronic colitis in susceptible individuals. Susceptibility has an immunogenetic basis: defining this is essential if nematode therapy is to be successfully and safely targeted in inflammatory bowel disease (IBD). To validate a preclinical mouse model we phenotyped the response to Trichuris muris in mice. We determined colonic transcriptional activity in naïve and infected mice and linked differential gene expression to mechanistic pathways. METHODS: T. muris-infected resistant (BALB/c) and susceptible (AKR) mice were studied to a chronic colitic timepoint (day 35). Colonic genome-wide expression was performed by microarray. Significant transcriptional changes were analyzed by cluster and gene ontology filtering and KEGG pathway mapping. RESULTS: Day 35 infected AKR displayed chronic diarrhea, weight loss, and transmural colonic inflammation; BALB/c remained asymptomatic, cleared the infection, and demonstrated normal histology. Compared to BALB/c mice, infected AKR upregulated gene expression clusters were overrepresented by immune response, chemotaxis, and apoptosis pathways. Cellular/tissue homeostasis and tight junction pathways dominated downregulated AKR expression clusters. Infected AKR T-helper cell development/polarization markers demonstrated predominant T(H) 1/T(H) 17 transcriptional activity. Colitic AKR data mirrored established murine models and human colitis. CONCLUSIONS: T. muris infection in the mouse shows striking phenotypic and transcriptional similarities to widely used models of IBD and human IBD. This preclinical mouse model presents a platform to examine biological commonalities among chronic colitides. However, these data urge caution in untargeted therapeutic helminth use until risk/benefit in susceptible individuals is more fully understood.

Exploitation of the intestinal microflora by the parasitic nematode Trichuris muris.

The inhabitants of the mammalian gut are not always relatively benign commensal bacteria but may also include larger and more parasitic organisms, such as worms and protozoa. At some level, all these organisms are capable of interacting with each other. We found that successful establishment of the chronically infecting parasitic nematode Trichuris muris in the large intestine of mice is dependent on microflora and coincident with modulation of the host immune response. By reducing the number of bacteria in the host animal, we significantly reduced the number of hatched T. muris eggs. Critical interactions between bacteria (microflora) and parasites (macrofauna) introduced a new dynamic to the intestinal niche, which has fundamental implications for our current concepts of intestinal homeostasis and regulation of immunity.