Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes.

CD4+ effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic Teff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (TH) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as TH markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ.

The Gastrointestinal Helminth Heligmosomoides bakeri Suppresses Inflammation in a Model of Contact Hypersensitivity.

Helminths regulate host immune responses to ensure their own long-term survival. Numerous studies have demonstrated that these helminth-induced regulatory mechanisms can also limit host inflammatory responses in several disease models. We used the Heligmosomoides bakeri (Hb) infection model (also known as H. polygyrus or H. polygyrus bakeri in the literature) to test whether such immune regulation affects skin inflammatory responses induced by the model contact sensitiser dibutyl phthalate fluorescein isothiocynate (DBP-FITC). Skin lysates from DBP-FITC-sensitized, Hb-infected mice produced less neutrophil specific chemokines and had significantly reduced levels of skin thickening and cellular inflammatory responses in tissue and draining lymph nodes (LNs) compared to uninfected mice. Hb-induced suppression did not appear to be mediated by regulatory T cells, nor was it due to impaired dendritic cell (DC) activity. Mice cleared of infection remained unresponsive to DBP-FITC sensitization indicating that suppression was not via the secretion of Hb-derived short-lived regulatory molecules, although long-term effects on cells cannot be ruled out. Importantly, similar helminth-induced suppression of inflammation was also seen in the draining LN after intradermal injection of the ubiquitous allergen house dust mite (HDM). These findings demonstrate that Hb infection attenuates skin inflammatory responses by suppressing chemokine production and recruitment of innate cells. These findings further contribute to the growing body of evidence that helminth infection can modulate inflammatory and allergic responses via a number of mechanisms with potential to be exploited in therapeutic and preventative strategies in the future.

Evidence for genes controlling resistance to Heligmosomoides bakeri on mouse chromosome 1.

Resistance to infections with Heligmosomoides bakeri is associated with a significant quantitative trait locus (QTL-Hbnr1) on mouse chromosome 1 (MMU1). We exploited recombinant mice, with a segment of MMU1 from susceptible C57Bl/10 mice introgressed onto MMU1 in intermediate responder NOD mice (strains 1094 and 6109). BALB/c (intermediate responder) and C57Bl/6 mice (poor responder) were included as control strains and strain 1098 (B10 alleles on MMU3) as NOD controls. BALB/c mice resisted infection rapidly and C57Bl/6 accumulated heavy worm burdens. Fecal egg counts dropped by weeks 10-11 in strain 1098, but strains 1094 and 6109 continued to produce eggs, harbouring more worms when autopsied (day 77). PubMed search identified 3 genes (Ctla4, Cd28, Icos) as associated with 'Heligmosomoides' in the B10 insert. Single nucleotide polymorphism (SNP) differences in Ctla4 could be responsible for regulatory changes in gene function, and a SNP within a splice site in Cd28 could have an impact on function, but no polymorphisms with predicted effects on function were found in Icos. Therefore, one or more genes encoded in the B10 insert into NOD mice contribute to the response phenotype, narrowing down the search for genes underlying the H. bakeri resistance QTL, and suggest Cd28 and Ctla4 as candidate genes.

Basophil-mediated protection against gastrointestinal helminths requires IgE-induced cytokine secretion.

Basophils orchestrate protection against reinfections with gastrointestinal helminths and ticks, but the underlying mechanisms remain elusive. We investigated the role of Fc receptors on basophils, the antibody isotypes IgG1 and IgE, and basophil-derived IL-4/IL-13 during challenge infections with Heligmosomoides polygyrus and Nippostrongylus brasiliensis. Using mixed bone marrow chimeras, we found that activating Fc receptors on basophils were required for protective immunity but not for regulation of basophil homeostasis. Furthermore, rapid worm expulsion was impaired in IgE-deficient but not in IgG1-deficient mice. Basophils promoted the recruitment of other effector cells into the small intestine and induced expression of the antihelminthic proteins resistin-like molecule ß and mucin 5ac. Selective deletion of IL-4/IL-13 in basophils resulted in impaired worm expulsion. Collectively, our results indicate that IgE-mediated activation of basophils and the release of basophil-derived IL-4/IL-13 are critical steps in protective immunity against helminths. Therefore, development of effective vaccines against helminths should consider boosting the IL-4/IgE/basophil axis of the immune system.

LPS and inactivated Propionibacterium acnes elicit a partially protective response in primary infections of Heligmosomoides polygyrus.

Intestinal helminth infections are common and of paramount economic importance in domestic animals. Available chemotherapy is limited and anthelmintic resistance is widespread in some hosts. This scenario favors the exploration of alternative methods of control, among them immune modulators. The effect of Escherichia coli LPS+Propionibacterium acnes on a primary infection of Heligmosomoides polygyrus (Trichostongyloidea) in mice has been tested. Nematode infection induced a rise of specific IgG1, both serum and intestinal, and a significant reduction in the unspecific (ConA) lymphoproliferative response. Treatment with the immune modulator (days -2, 0, 7 and 14 post infection) elicited an apparent delay of larval intramucosal development. Moreover cumulative nematode egg shedding in treated mice was significantly lower (p=0.0041). Preliminary results point toward the interest of immune modulators to control intestinal helminths.

Protein deficiency alters impact of intestinal nematode infection on intestinal, visceral and lymphoid organ histopathology in lactating mice.

Protein deficiency impairs local and systemic immune responses to Heligmosomoides bakeri infection but little is known about their individual and interactive impacts on tissue architecture of maternal lymphoid (thymus, spleen) and visceral (small intestine, kidney, liver, pancreas) organs during the demanding period of lactation. Using a 2 × 2 factorial design, pregnant CD1 mice were fed a 24% protein sufficient (PS) or a 6% protein deficient (PD) isoenergetic diet beginning on day 14 of pregnancy and were infected with 100 H. bakeri larvae four times or exposed to four sham infections. On day 20 of lactation, maternal organs were examined histologically and serum analytes were assayed as indicators of organ function. The absence of villus atrophy in response to infection was associated with increased crypt depth and infiltration of mast cells and eosinophils but only in lactating dams fed adequate protein. Infection-induced lobular liver inflammation was reduced in PD dams, however, abnormalities in the kidney caused by protein deficiency were absent in infected dams. Bilirubin and creatinine were highest in PD infected mice. Infection-induced splenomegaly was not due to an increase in the lymphoid compartment of the spleen. During lactation, infection and protein deficiency have interactive effects on extra-intestinal pathologies.

The immunology and ecology of co-infection.

It's a wormy world. All natural vertebrate populations are subject to infection and re-infection with helminth parasites (Stoll 1947). Even in humans, around one billion people in developing nations are infected by one or several of a range of helminth parasites (Lustigman et al. 2012). Infection by worms is therefore the norm and is reflected in vertebrate immune responses. Thus, there is probably little point in generating an inflammatory response to clear every last worm, with ensuing collateral damage to our own tissue, when rapid re-infection from the environment by another worm is pretty much assured. Instead, the vertebrate immune system modifies its response to worms, controlling (but not always clearing) these infections and at the same time limiting damage to host tissue caused by inflammatory immune responses (Jackson et al. 2009). The immune system, however, has to fight battles on several fronts and, while fighting a prolonged war of attrition against helminth parasites, it also has to protect against periodic invasion by bacteria, where a rapid response to kill invading microbes before they spread is essential. In this issue of Molecular Ecology, Friberg et al. (2013) ask what effect worm infections have on a host's ability to mount antimicrobial responses.

Selenium status alters the immune response and expulsion of adult Heligmosomoides bakeri worms in mice.

Heligmosomoides bakeri is a nematode with parasitic development exclusively in the small intestine of infected mice that induces a potent STAT6-dependent Th2 immune response. We previously demonstrated that host protective expulsion of adult H. bakeri worms from a challenge infection was delayed in selenium (Se)-deficient mice. In order to explore mechanisms associated with the delayed expulsion, 3-week-old female BALB/c mice were placed on a torula yeast-based diet with or without 0.2 ppm Se, and after 5 weeks, they were inoculated with H. bakeri infective third-stage larvae (L3s). Two weeks after inoculation, the mice were treated with an anthelmintic and then rested, reinoculated with L3s, and evaluated at various times after reinoculation. Analysis of gene expression in parasite-induced cysts and surrounding tissue isolated from the intestine of infected mice showed that the local-tissue Th2 response was decreased in Se-deficient mice compared to that in Se-adequate mice. In addition, adult worms recovered from Se-deficient mice had higher ATP levels than worms from Se-adequate mice, indicating greater metabolic activity in the face of a suboptimal Se-dependent local immune response. Notably, the process of worm expulsion was restored within 2 to 4 days after feeding a Se-adequate diet to Se-deficient mice. Expulsion was associated with an increased local expression of Th2-associated genes in the small intestine, intestinal glutathione peroxidase activity, secreted Relm-ß protein, anti-H. bakeri IgG1 production, and reduced worm fecundity and ATP-dependent metabolic activity.

Macroparasites at peripheral sites of infection are major and dynamic modifiers of systemic antimicrobial pattern recognition responses.

Immune defences and the maintenance of immunological homeostasis in the face of pathogenic and commensal microbial exposures are channelled by innate antimicrobial pattern recognition receptors (PRRs) such as toll-like receptors (TLRs). Whilst PRR-mediated response programmes are the result of long-term host-pathogen or host-commensal co-evolutionary dynamics involving microbes, an additional possibility is that macroparasitic co-infections may be a significant modifier of such interactions. We demonstrate experimentally that macroparasites (the model gastrointestinal nematode, Heligmosomoides) at peripheral sites of infection cause substantial alteration of the expression and function of TLRs at a systemic level (in cultured splenocytes), predominantly up-regulating TLR2, TLR4 and TLR9-mediated cytokine responses at times of high standing worm burdens. We consistently observed such effects in BALB/c and C57BL/6 mice under single-pulse and trickle exposures to Heligmosomoides larvae and in SWR and CBA mice under single-pulse exposures. A complementary long-term survey of TLR2-mediated tumour necrosis factor-alpha responses in wild wood mice (Apodemus sylvaticus) was consistent with substantial effects of macroparasites under some environmental conditions. A general pattern, though, was for the associations of macroparasites with TLR function to be temporally dynamic and context-dependent: varying with different conditions of infection exposure in the field and laboratory and with host genetic strain in the laboratory. These results are compelling evidence that macroparasites are a major and dynamic modifier of systemic innate antimicrobial responsiveness in naturally occurring mammals and thus likely to be an important influence on the interaction between microbial exposures and the immune system.

Maternal protein deficiency during a gastrointestinal nematode infection alters developmental profile of lymphocyte populations and selected cytokines in neonatal mice.

Neonatal immune development begins in pregnancy and continues into lactation and may be affected by maternal diet. We investigated the possibility that maternal protein deficiency (PD) during a chronic gastrointestinal (GI) nematode infection could impair neonatal immune development. Beginning on d 14 of pregnancy, mice were fed protein-sufficient (PS; 24%) or protein-deficient (PD; 6%) isoenergetic diets and were infected weekly with either 0 (sham) or 100 Heligmosomoides bakeri larvae. Pups were killed on d 2, 7, 14, and d 21 and dams on d 20 of lactation. Lymphoid organs were weighed. Cytokine concentration in maternal and pup serum and in milk from pup stomachs and lymphoid cell populations in pup spleen and thymus were determined using luminex and flow cytometry, respectively. GI nematode infection increased Th2 cytokines (IL-4, IL-5, IL-13), IL-2, IL-10, and eotaxin in serum of dams whereas PD reduced IL-4 and IL-13. The lower IL-13 in PD dams was associated with increased fecal egg output and worm burdens. Maternal PD increased vascular endothelial growth factor in pup milk and eotaxin in pup serum. Maternal infection increased eotaxin in pup serum. Evidence of impaired neonatal immune development included reduced lymphoid organ mass in pups associated with both maternal infection and PD and increased percentage of T cells and T:B cell ratio in the spleen associated with maternal PD. Findings suggest that increases in specific proinflammatory cytokines as a result of the combination of infection and dietary PD in dams can impair splenic immune development in offspring.

Heligmosomoides bakeri: a model for exploring the biology and genetics of resistance to chronic gastrointestinal nematode infections.

The intestinal nematode Heligmosomoides bakeri has undergone 2 name changes during the last 4 decades. Originally, the name conferred on the organism in the early 20th century was Nematospiroides dubius, but this was dropped in favour of Heligmosomoides polygyrus, and then more recently H. bakeri, to distinguish it from a closely related parasite commonly found in wood mice in Europe. H. bakeri typically causes long-lasting infections in mice and in this respect it has been an invaluable laboratory model of chronic intestinal nematode infections. Resistance to H. bakeri is a dominant trait and is controlled by genes both within and outside the MHC. More recently, a significant QTL has been identified on chromosome 1, although the identity of the underlying genes is not yet known. Other QTL for resistance traits and for the accompanying immune responses were also defined, indicating that resistance to H. bakeri is a highly polygenic phenomenon. Hence marker-assisted breeding programmes aiming to improve resistance to GI nematodes in breeds of domestic livestock will need to be highly selective, focussing on genes that confer the greatest proportion of overall genetic resistance, whilst leaving livestock well-equipped genetically to cope with other types of pathogens and preserving important production traits.

Cryptosporidium parvum: the course of Cryptosporidium parvum infection in C57BL/6 mice co-infected with the nematode Heligmosomoides bakeri.

The effects of Heligmosomoides bakeri infection on the course of a concurrent Cryptosporidium parvum infection were studied in C57BL/6 mice. Mice were initially infected with 80 L(3) of H. bakeri and then challenged with 10(4) oocysts of C. parvum, administered during the patent period of the nematode infection (28 day post H. bakeri infection). The number of C. parvum oocysts excreted in the feces and the number of adult H. bakeri in the small intestine were monitored during the experiment. Concurrent H. bakeri infection resulted in a prolonged course of infection with C. parvum. The intensities of both parasite infections were higher in co-infections. We also investigated the cellular immune response at 14 and 42 days post infection C. parvum. During infection with C. parvum there was an increase in production of IFN-gamma and IL-12 but co-infection with H. bakeri inhibited IFN-gamma secretion. The present study is the first to demonstrate that infection with H. bakeri markedly exacerbates the intensity of a concurrent C. parvum infection in laboratory mice and also affects immune effectors mechanisms in co-infection with H. bakeri.

Granulomatous inflammation during Heligmosomoides polygyrus primary infections in FVB mice.

Host responses to primary infections with Heligmosomoides polygyrus were studied in fast responding FVB mice (H-2(q)). Pathological changes in the intestinal mucosa, mesenteric lymph nodes and spleen were examined. Features of the fast response were typical: low effectiveness of infection and limiting of parasite survival and egg production, with worm expulsion occurring about 60 days post-infection. The intestinal inflammatory response involved infiltration by different cells into the intestinal mucosa and granulomata formation. As is typical for intestinal nematode infection enteropathy, decreased villus:crypt ratio and hyperplasia of goblet and Paneth cells were also present. Reactions of the intestinal mucosa, mesenteric lymph nodes and spleen increased over time post-infection and after worm expulsion. Enteropathy may help worm expulsion by creating an unfavourable environment for H. polygyrus. The implications of these findings and the potential role of intestinal intraepithelial lymphocytes in the pathogenesis of generated lesions are discussed.

The role of IL-4 in Heligmosomoides polygyrus-induced alterations in murine intestinal epithelial cell function.

IL-4 and IL-13 promote gastrointestinal worm expulsion, at least in part, through effects on nonlymphoid cells, such as intestinal epithelial cells. The role of IL-4/IL-13 in the regulation of intestinal epithelial function during Heligmosomoides polygyrus (Hp) infection was investigated in BALB/c mice infected with Hp or treated with a long-lasting formulation of recombinant mouse IL-4/alphaIL-4 complexes (IL-4C) for 7 days. Separate groups of BALB/c mice were drug-cured of initial infection and later reinfected and treated with anti-IL-4R mAb, an antagonist of IL-4 and IL-13 receptor binding, or with a control mAb. Segments of jejunum were mounted in Ussing chambers, and short circuit current responses to acetylcholine, histamine, serotonin, PGE2, and glucose were determined. Although only modest changes in epithelial cell function were observed during primary Hp infection, IL-4C or a secondary Hp infection each induced more dramatic changes, including increased mucosal permeability, reduced sodium-linked glucose absorption, and increased Cl- secretory response to PGE2. Some, but not all, effects of IL-4C and Hp infection were dependent on enteric nerves. Hp-induced changes in epithelial function were attenuated or prevented by anti-IL-4R mAb. Thus, IL-4/IL-13 mediate many of the effects of Hp infection on intestinal epithelial cell function and do so both through direct effects on epithelial cells and through indirect, enteric nerve-mediated prosecretory effects. These immune system-independent effector functions of IL-4/IL-13 may be important for host protection against gastrointestinal nematodes.

Role of B7 signaling in the differentiation of naive CD4+ T cells to effector interleukin-4-producing T helper cells.

Signaling through the T cell receptor must be accompanied by costimulatory signals for the differentiation of naive T cells to cytokine-producing effector T helper cells. The costimulatory signal through CD28 is required for T cell activation resulting in increased interleukin (IL)-2 production in vitro, but its role in the production of IL-4 and in the in vivo response is still unclear. We have examined the effects of blocking CTLA-4 (the CD28 homologue) ligand interactions on the in vivo development of IL-4-producing T helper effector cells during a primary mucosal immune response to the nematode parasite Heligmosomoides polygyrus and during a primary systemic immune response to immunogenic anti-IgD antibodies. Our results demonstrate that CD28 and/or CTLA-4 signaling is required for T cell priming leading to IL-4 cytokine production, B cell activation, and IgE secretion during both immune responses, suggesting that other signaling molecules do not substitute for these molecules in either of these two different immune responses. Furthermore, the CD28 ligands, B7-1 and B7-2, can substitute for each other in providing the required T cell costimulatory ligand interactions during the primary immune response to H. polygyrus. In contrast, memory T cells during the challenge immune response do not require CD28/CTLA-4 ligand interactions for IL-4 production and T helper effector function.

CTLA-4 ligands are required to induce an in vivo interleukin 4 response to a gastrointestinal nematode parasite.

The costimulatory signal provided to T cells through CTLA-4-ligand interactions is required for T cell activation resulting in increased interleukin 2 (IL-2) production in vitro, but its role in the production of IL-4 and other cytokines is unclear and few in vivo studies have been performed to confirm results of in vitro experiments. We have examined the in vivo effects of blocking CTLA-4 ligands on the T helper cell 2 (Th2)-associated mucosal immune response that follows oral infection of mice with the nematode parasite, Heligmosomoides polygyrus. CTLA-4Ig administration inhibited H. polygyrus-induced increases in mesenteric lymph node (MLN) B cell major histocompatibility complex class II expression and size and T cell-derived IL-4 gene expression. In addition, CTLA-4 immunoglobulin (Ig) partially blocked increased IL-3, IL-5, and IL-9 cytokine gene expression in Peyer's patch (PP) and MLN 8 d after primary inoculation of mice with the parasite. Increases in the number of IL-4- but not IL-5-secreting cells were also inhibited by CTLA-4Ig. H. polygyrus-induced elevations in serum IgE levels but not blood eosinophils, were markedly inhibited by CTLA-4Ig. These results suggest that stimulation of CD28 and/or CTLA-4 is required for T cell priming leading to IL-4 cytokine production, B cell activation, and IgE secretion during a Th2-like, mucosal immune response to a nematode parasite.

Interleukin 4 is important in protective immunity to a gastrointestinal nematode infection in mice.

Parasitic helminths typically induce components of immediate-type hypersensitivity, including elevated serum IgE, eosinophilia, and mucosal mast cells. These responses are T-cell-dependent and associated with rapid expulsion of parasitic worms from a sensitized host; existing experimental systems have failed to define the precise role of cytokines in these responses. We report here that anti-interleukin 4 or anti-interleukin 4 receptor antibodies block the polyclonal IgE response to a parasitic nematode, Heligmosomoides polygyrus, and abrogate protective immunity to the infection. In contrast, anti-interleukin 5 antibody prevented H. polygyrus-induced eosinophilia but did not prevent protection. These data provide evidence that a specific cytokine affects the physiology and survival of a parasitic nematode in the host.

Immunological relationships during primary infection with Heligmosomoides polygyrus (Nematospiroides dubius): expulsion of adult worms from fast responder syngeneic and hybrid strains of mice.

The time-course of low and high intensity primary infections with Heligmosomoides polygyrus was monitored in SJL and SWR mice, both of which usually expel worms within 7 weeks of larval administration. Worm expulsion in these strains was not dependent on the intensity of infection, with low and high intensity worm burdens being lost within the same period of time. The ability to expel worms rapidly was inherited in a dominant manner in F1 offspring of SJL or SWR mice mated with C57Bl10 mice; the latter being a strain in which no loss of worms was evident within 10 weeks of infection. However, neither (SJL x C57Bl10)F1 nor (SWR x C57Bl10)F1 mice expelled worms as rapidly as the parental SJL and SWR strains. (SWR x B10G)F1 [H-2q] mice eliminated worms faster than (SWR x C57Bl10)F1 [H-2bq], suggesting that the b haplotype had a moderating influence on the expulsion process. In fact (SWR x B10G)F1 mice showed a significant reduction in worm burdens by week 4 but by weeks 6-8 the rate of worm loss had slowed considerably. In contrast, SJL and SWR mice, whilst initiating rejection slightly later, (after week 4) expelled all worms within the following 2 weeks. Thus two distinct patterns of response were observed among the fast responder strains as exemplified by SWR and SJL mice on the one hand and (SWR x B10G)F1 on the other. Our results support the hypothesis that the course of a primary infection with H. polygyrus is influenced by multiple host gene loci, some of which are encoded within the MHC. SJL and SWR mice probably have similar if not identical gene combinations at loci which determine a fast responder phenotype, distinguishing them from the other mouse strains which have been studied.

Excretory-secretory antigens from adult Nematospiroides dubius.

Adult Nematospiroides dubius excretory-secretory (ES) products were collected from worms cultured in vitro, radiolabelled and separated by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). The components were characterized and assessed for molecular weight (MW) after autoradiography and gel staining, for antigenicity in immunoblots, for sensitivity to protease enzymes, and for carbohydrate content. ES contained at least 18 denatured components from MW less than 20,000 to greater than 200,000. At least one of the surface proteins of adults was found with the ES antigens recovered when adults were cultured in vitro. Molecules with MW 200,000, 78,000 and 60,000 were glycoproteins and reacted with immune mouse serum in Western blots. The dominant ES, MW 60,000 component stained with periodic acid-Schiff (PAS), bound lectins with affinity for D-mannose, and was resistant to peptic and tryptic but not V8 protease digestion.