A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model.

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

Effects of Raising or Lowering Molybdenum Status on Outcome of Acute Infection with Nippostrongylus brasiliensis in Mature Rats.

Molybdate (Mo+) supplements can suppress or enhance nematode infections in ruminants, depending on exposure level, but there have been no investigations in non-ruminants. Three groups of 16 mature rats were each fed a commercial diet and given Mo+ (10 mg Mo/l), tungstate (a molybdenum [Mo] antagonist) (MoO4, 350 mg W/l) or no supplement (C) via drinking water for 40 days before acute infection with 3,600 Nippostrongylus brasiliensis larvae. Group Mo- also received allopurinol (1 g/l), a molybdenoenzyme inhibitor, from 4 days post infection (dpi). Subgroups of four rats from each group were killed at 7-14 dpi. A group of six rats was left untreated and uninfected and subgroups killed 10 or 12 dpi. Infection reduced intakes of food and water but impacts were greatest in group Mo-. Median worm counts in groups C, Mo- and Mo+ were 900, 941 and 510, respectively, at 7 dpi and 9, 40 and 0 (P = 0.05) at 10 dpi. Median faecal egg counts were consistently lowest in group Mo+. Worm weight was reduced (P <0.05), worm tissue protease increased and superoxide dismutase activities increased in worm (P < 0.01) and host duodenal homogenates (P < 0.01) from group Mo+. In group Mo-, liver Mo concentration decreased, duodenal xanthine oxidoreductase activity (DXOR) became totally inhibited and plasma uric acid was barely detectable at 10 dpi. Plasma mast cell protease activity and duodenal malonyldialdehyde concentrations, markers of inflammation, were increased by nematode infection (P <0.001) but unaffected by water treatments. Liver Mo, liver copper (Cu) and plasma Cu concentrations were increased in group Mo+ and plasma Cu concentration was increased in group Mo- suggesting systemic exposure to partially thiolated MoO4 and WO4. Supplementary MoO4 impaired larval establishment and changed parasite biochemistry without affecting the inflammatory response to infection but may have required partial thiolation to do so. Rats did not rely on DXOR activity to expel N. brasiliensis.

Loss of protein tyrosine phosphatase non-receptor type 2 reduces IL-4-driven alternative macrophage activation.

Macrophages are a heterogeneous population of innate immune cells that are often divided into two major subsets: classically activated, typically pro-inflammatory (M1) macrophages that mediate host defense, and alternatively activated, tolerance-inducing (M2) macrophages that exert homeostatic and tissue-regenerative functions. Disturbed macrophage function/differentiation results either in inadequate, excessive immune activation or in a failure to induce efficient protective immune responses against pathogens. Loss-of-function variants in protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with chronic inflammatory disorders, but the effect of macrophage-intrinsic PTPN2 loss is still poorly understood. Here we report that PTPN2-deficient macrophages fail to acquire an alternatively activated/M2 phenotype. This was the consequence of reduced IL-6 receptor expression and a failure to induce IL-4 receptor in response to IL-6, resulting in an inability to respond to the key M2-inducing cytokine IL-4. Ultimately, failure to adequately respond to IL-6 and IL-4 resulted in increased levels of M1 macrophage marker expression in vitro and exacerbated lung inflammation upon infection with Nippostrongylus brasiliensis in vivo. These results demonstrate that PTPN2 loss interferes with the ability of macrophages to adequately respond to inflammatory stimuli and might explain the increased susceptibility of PTPN2 loss-of-function carriers to developing inflammatory diseases.

Intestinal epithelial tuft cell induction is negated by a murine helminth and its secreted products.

Helminth parasites are adept manipulators of the immune system, using multiple strategies to evade the host type 2 response. In the intestinal niche, the epithelium is crucial for initiating type 2 immunity via tuft cells, which together with goblet cells expand dramatically in response to the type 2 cytokines IL-4 and IL-13. However, it is not known whether helminths modulate these epithelial cell populations. In vitro, using small intestinal organoids, we found that excretory/secretory products (HpES) from Heligmosomoides polygyrus blocked the effects of IL-4/13, inhibiting tuft and goblet cell gene expression and expansion, and inducing spheroid growth characteristic of fetal epithelium and homeostatic repair. Similar outcomes were seen in organoids exposed to parasite larvae. In vivo, H. polygyrus infection inhibited tuft cell responses to heterologous Nippostrongylus brasiliensis infection or succinate, and HpES also reduced succinate-stimulated tuft cell expansion. Our results demonstrate that helminth parasites reshape their intestinal environment in a novel strategy for undermining the host protective response.

Up-regulation of gasdermin C in mouse small intestine is associated with lytic cell death in enterocytes in worm-induced type 2 immunity.

"Taste-like" tuft cells in the intestine trigger type 2 immunity in response to worm infection. The secretion of interleukin-13 (IL-13) from type 2 innate lymphoid cells (ILC2) represents a key step in the tuft cell-ILC2 cell-intestinal epithelial cell circuit that drives the clearance of worms from the gut via type 2 immune responses. Hallmark features of type 2 responses include tissue remodeling, such as tuft and goblet cell expansion, and villus atrophy, yet it remains unclear if additional molecular changes in the gut epithelium facilitate the clearance of worms from the gut. Using gut organoids, we demonstrated that IL-4 and IL-13, two type 2 cytokines with similar functions, not only induced the classical type 2 responses (e.g., tuft cell expansion) but also drastically up-regulated the expression of gasdermin C genes (Gsdmcs). Using an in vivo worm-induced type 2 immunity model, we confirmed the up-regulation of Gsdmcs in Nippostrongylus brasiliensis-infected wild-type C57BL/6 mice. Consistent with gasdermin family members being principal effectors of pyroptosis, overexpression of Gsdmc2 in human embryonic kidney 293 (HEK293) cells triggered pyroptosis and lytic cell death. Moreover, in intestinal organoids treated with IL-4 or IL-13, or in wild-type mice infected with N. brasiliensis, lytic cell death increased, which may account for villus atrophy observed in worm-infected mice. Thus, we propose that the up-regulated Gsdmc family may be major effectors for type 2 responses in the gut and that Gsdmc-mediated pyroptosis may provide a conduit for the release of antiparasitic factors from enterocytes to facilitate the clearance of worms.

Tumor suppressor p53 regulates intestinal type 2 immunity.

The role of p53 in tumor suppression has been extensively studied and well-established. However, the role of p53 in parasitic infections and the intestinal type 2 immunity is unclear. Here, we report that p53 is crucial for intestinal type 2 immunity in response to the infection of parasites, such as Tritrichomonas muris and Nippostrongylus brasiliensis. Mechanistically, p53 plays a critical role in the activation of the tuft cell-IL-25-type 2 innate lymphoid cell circuit, partly via transcriptional regulation of Lrmp in tuft cells. Lrmp modulates Ca2+ influx and IL-25 release, which are critical triggers of type 2 innate lymphoid cell response. Our results thus reveal a previously unrecognized function of p53 in regulating intestinal type 2 immunity to protect against parasitic infections, highlighting the role of p53 as a guardian of immune integrity.

Basophils prime group 2 innate lymphoid cells for neuropeptide-mediated inhibition.

Type 2 cytokine responses promote parasitic immunity and initiate tissue repair; however, they can also result in immunopathologies when not properly restricted. Although basophilia is recognized as a common feature of type 2 inflammation, the roles basophils play in regulating these responses are unknown. Here, we demonstrate that helminth-induced group 2 innate lymphoid cell (ILC2) responses are exaggerated in the absence of basophils, resulting in increased inflammation and diminished lung function. Additionally, we show that ILC2s from basophil-depleted mice express reduced amounts of the receptor for the neuropeptide neuromedin B (NMB). Critically, NMB stimulation inhibited ILC2 responses from control but not basophil-depleted mice, and basophils were sufficient to directly enhance NMB receptor expression on ILC2s. These studies suggest that basophils prime ILC2s to respond to neuron-derived signals necessary to maintain tissue integrity. Further, these data provide mechanistic insight into the functions of basophils and identify NMB as a potent inhibitor of type 2 inflammation.

IL-17A both initiates, via IFNγ suppression, and limits the pulmonary type-2 immune response to nematode infection.

Nippostrongylus brasiliensis is a well-defined model of type-2 immunity but the early lung-migrating phase is dominated by innate IL-17A production. In this study, we confirm previous observations that Il17a-KO mice infected with N. brasiliensis exhibit an impaired type-2 immune response. Transcriptional profiling of the lung on day 2 of N. brasiliensis infection revealed an increased Ifng signature in Il17a-KO mice confirmed by enhanced IFNγ protein production in lung lymphocyte populations. Depletion of early IFNγ rescued type-2 immune responses in the Il17a-KO mice demonstrating that IL-17A-mediated suppression of IFNγ promotes type-2 immunity. Notably, later in infection, once the type-2 response was established, IL-17A limited the magnitude of the type-2 response. IL-17A regulation of type-2 immunity was lung-specific and infection with Trichuris muris revealed that IL-17A promotes a type-2 immune response in the lung even when infection is restricted to the intestine. Together our data reveal IL-17A as a major regulator of pulmonary type-2 immunity such that IL-17A supports early development of a protective type-2 response by suppression of IFNγ but subsequently limits excessive type-2 responses. A failure of this feedback loop may contribute to conditions such as severe asthma, characterised by combined elevation of IL-17 and type-2 cytokines.

Tuft-Cell-Derived Leukotrienes Drive Rapid Anti-helminth Immunity in the Small Intestine but Are Dispensable for Anti-protist Immunity.

Helminths, allergens, and certain protists induce type 2 immune responses, but the underlying mechanisms of immune activation remain poorly understood. In the small intestine, chemosensing by epithelial tuft cells results in the activation of group 2 innate lymphoid cells (ILC2s), which subsequently drive increased tuft cell frequency. This feedforward circuit is essential for intestinal remodeling and helminth clearance. ILC2 activation requires tuft-cell-derived interleukin-25 (IL-25), but whether additional signals regulate the circuit is unclear. Here, we show that tuft cells secrete cysteinyl leukotrienes (cysLTs) to rapidly activate type 2 immunity following chemosensing of helminth infection. CysLTs cooperate with IL-25 to activate ILC2s, and tuft-cell-specific ablation of leukotriene synthesis attenuates type 2 immunity and delays helminth clearance. Conversely, cysLTs are dispensable for the tuft cell response induced by intestinal protists. Our findings identify an additional tuft cell effector function and suggest context-specific regulation of tuft-ILC2 circuits within the small intestine.

Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven Parameters by Standard Confocal Microscopy.

Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced. Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc. Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodies Support Protocol 1: Antibody titration protocol Support Protocol 2: Spillover optimization protocol.

Neuropeptide CGRP Limits Group 2 Innate Lymphoid Cell Responses and Constrains Type 2 Inflammation.

Innate lymphocytes maintain tissue homeostasis at mucosal barriers, with group 2 innate lymphoid cells (ILC2s) producing type 2 cytokines and controlling helminth infection. While the molecular understanding of ILC2 responses has advanced, the complexity of microenvironmental factors impacting ILC2s is becoming increasingly apparent. Herein, we used single-cell analysis to explore the diversity of gene expression among lung lymphocytes during helminth infection. Following infection, we identified a subset of ILC2s that preferentially expressed Il5-encoding interleukin (IL)-5, together with Calca-encoding calcitonin gene-related peptide (CGRP) and its cognate receptor components. CGRP in concert with IL-33 and neuromedin U (NMU) supported IL-5 but constrained IL-13 expression and ILC2 proliferation. Without CGRP signaling, ILC2 responses and worm expulsion were enhanced. Collectively, these data point to CGRP as a context-dependent negative regulatory factor that shapes innate lymphocyte responses to alarmins and neuropeptides during type 2 innate immune responses.

The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation.

Fine control of macrophage activation is needed to prevent inflammatory disease, particularly at barrier sites such as the lungs. However, the dominant mechanisms that regulate the activation of pulmonary macrophages during inflammation are poorly understood. We found that alveolar macrophages (AlvMs) were much less able to respond to the canonical type 2 cytokine IL-4, which underpins allergic disease and parasitic worm infections, than macrophages from lung tissue or the peritoneal cavity. We found that the hyporesponsiveness of AlvMs to IL-4 depended upon the lung environment but was independent of the host microbiota or the lung extracellular matrix components surfactant protein D (SP-D) and mucin 5b (Muc5b). AlvMs showed severely dysregulated metabolism relative to that of cavity macrophages. After removal from the lungs, AlvMs regained responsiveness to IL-4 in a glycolysis-dependent manner. Thus, impaired glycolysis in the pulmonary niche regulates AlvM responsiveness during type 2 inflammation.

Sinistral, Dextral, and Mixed Coiling Patterns Observed in Heligmonellid Nematodes (Trichostrongyloidea).

Coiling patterns of heligmonellid nematodes were examined for 520, 208, and 33 individuals of Nippostrongylus brasiliensis, Orientostrongylus tenorai, and Sabanema sp., respectively, collected from murine rodents of Indonesia. Besides typical sinistral coiling, complete dextral coiling was found in 3.3% of N. brasiliensis and 12.1% of Sabanema sp. Mixed coiling with partial sinistral and dextral patterns was also observed in 38.8% of N. brasiliensis, 60.7% of Sabanema sp., and 3.4% of O. tenorai. In dextral coils, the left ventral area with large ridges was located inside as in sinistral coils, keeping the ability to cling to intestinal villi. The cuticular dilatation at left to left dorsal area was located caudally in sinistral coils but rostrally in dextral coils. Presence of mixed coiling indicates that the coiling patterns can change. As the transition of coiling pattern accompanies a change in direction of coil axis, it is surmised that the dextral coiling may be chosen when a worm leaves a villus to move to another villus.

Intestinal helminth infection promotes IL-5- and CD4+ T cell-dependent immunity in the lung against migrating parasites.

The ability of helminths to manipulate the immune system of their hosts to ensure their own survival is often credited with affecting responses to other pathogens. We undertook co-infection experiments in mice to determine how infection with the intestinal helminth Heligmosomoides polygyrus affected the parasitological, immunological and physiological outcomes of a primary infection with a distinct species of helminth; the lung migratory parasite Nippostrongylus brasiliensis. We found that migrating N. brasiliensis larvae were killed in the lungs of H. polygyrus-infected mice by a process involving IL-33-activated CD4+ T cells that released IL-5 and recruited activated eosinophils. The lung pathology normally associated with N. brasiliensis larval migration was also reduced. Importantly, lung immunity remained intact in mice cleared of prior H. polygyrus infection and also occurred during infection with another entirely enteric helminth, Trichuris muris. This study identifies a cross-mucosal immune mechanism by which intestinal helminths may protect their hosts against co-infection by a different parasite at a distal site, via circulation of activated CD4+ T cells that can be triggered to release effector cytokines and mount inflammatory responses by tissue damage-associated alarmins, such as IL-33.

Selective expression of constitutively activated STAT6 in intestinal epithelial cells promotes differentiation of secretory cells and protection against helminths.

Intestinal epithelial cells (IECs) constitute an important barrier between host and pathogen. Immune mechanisms that provide protection against gastrointestinal helminths often require IL-4Rα-induced activation of STAT6-regulated genes in IECs. However, it is not known whether STAT6 activation in IECs enhances protective immunity against helminths. Furthermore, the regulation of proliferation and differentiation processes of the intestinal epithelium by IEC-intrinsic STAT6 signaling remains unclear. To address these questions, we generated mice with specific expression of a constitutively active version of STAT6 in IECs. These VillinCre_STAT6vt mice show accumulation of secretory IECs, increased proliferation of IECs and lengthening of the small intestine. They rapidly expelled Nippostrongylus brasiliensis worms even in the absence of T cells. Furthermore, primary infection with Heligmosomoides polygyrus resulted in larval trapping in the submucosa and the fecundity of adult worms was severely impaired. Our results reveal an important IEC-intrinsic role of STAT6-regulated genes for intestinal homeostasis and protective immunity against helminths.

B Cells Produce the Tissue-Protective Protein RELMα during Helminth Infection, which Inhibits IL-17 Expression and Limits Emphysema.

Emphysema results in destruction of alveolar walls and enlargement of lung airspaces and has been shown to develop during helminth infections through IL-4R-independent mechanisms. We examined whether interleukin 17A (IL-17A) may instead modulate development of emphysematous pathology in mice infected with the helminth parasite Nippostrongylus brasiliensis. We found that transient elevations in IL-17A shortly after helminth infection triggered subsequent emphysema that destroyed alveolar structures. Furthermore, lung B cells, activated through IL-4R signaling, inhibited early onset of emphysematous pathology. IL-10 and other regulatory cytokines typically associated with B regulatory cell function did not play a major role in this response. Instead, at early stages of the response, B cells produced high levels of the tissue-protective protein, Resistin-like molecule α (RELMα), which then downregulated IL-17A expression. These studies show that transient elevations in IL-17A trigger emphysema and reveal a helminth-induced immune regulatory mechanism that controls IL-17A and the severity of emphysema.

Nematode-Infected Mice Acquire Resistance to Subsequent Infection With Unrelated Nematode by Inducing Highly Responsive Group 2 Innate Lymphoid Cells in the Lung.

The immune responses against helminths have been investigated individually, and it is well-established that infected hosts develop an immunological memory to resist reinfection by the same pathogen. In contrast, it is poorly understood how the host immune system responds to subsequent infection by unrelated parasites after elimination of the first infection. We previously reported that infection of mice with Strongyloides venezuelensis induces the accumulation of group 2 innate lymphoid cells (ILC2s) in the lung. Here, we demonstrated that S. venezuelensis-experienced (Sv-exp) mice became significantly resistant against infection by Nippostrongylus brasiliensis. N. brasiliensis infection induced enhanced accumulation of ILC2s and eosinophils with increased expressions of mRNA for Th2 cytokines in the lungs of Sv-exp mice. The resistance was dependent on ILC2s, and eosinophils but not on CD4+ T cells. Furthermore, pulmonary ILC2s in Sv-exp mice acquired a highly responsive "trained" phenotype; in response to N. brasiliensis infection, they rapidly increased and produced IL-5 and IL-13, which in turn induced the early accumulation of eosinophils in the lungs. IL-33 was required for the accumulation of ILC2s and the resistance of mice against N. brasiliensis infection but insufficient for the induction of trained ILC2s. In conclusion, animals infected with one type of lung-migratory nematodes acquire a specific-antigen-independent resistance to another type of lung-migrating nematodes, providing animals with the capacity to protect against sequential infections with various lung-migratory nematodes.

MicroRNA-155 Protects Group 2 Innate Lymphoid Cells From Apoptosis to Promote Type-2 Immunity.

Group-2 innate lymphoid cells (ILC2) play critical roles in the initiation and maintenance of type-2 immune responses, predominantly through their production of the type-2 cytokines IL-5, IL-9, and IL-13. ILC2 are essential for the efficient elimination of helminth parasites, but also contribute to the detrimental type-2 immune responses that underlie diseases such as asthma and allergy. While several transcription factors have been identified that regulate the development and function of ILC2, less is known about the post-transcriptional mechanisms that regulate these processes. We identified micro-RNAs (miRNAs) that are co-ordinately regulated in ILC2 from mice exposed to two different stimuli, namely IL-33 "alarmin" administration or Nippostrongylus brasiliensis parasitic worm infection. miR-155 is upregulated in ILC2 in response to both stimuli and miR-155-/- mice had impaired IL-33-driven ILC2 responses. Using mixed bone marrow chimeras, we demonstrate that this deficit is intrinsic to ILC2 and that miR-155 protects ILC2 from apoptosis, while having little impact on ILC2 proliferation or cytokine production. These data reveal a subset of miRNAs that are regulated upon ILC2 activation and establish a specific role for miR-155 in regulating ILC2 survival following activation.

Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice.

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite-host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1ß, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.

Nippostrongylus brasiliensis infection leads to impaired reference memory and myeloid cell interference.

Hookworm infection is endemic in developing countries, leading to poor cognitive function-among other disruptions. In this study, the effects of Nippostrongylus brasiliensis infection (a murine model of Necator Americanus) on cognitive function were investigated. Though impaired cognition has been extensively reported, the exact domain of cognition affected is still unknown, hence requiring investigation. The objective of this study was to identify possible cognitive changes during Nippostrongylus brasiliensis infection in mice, using the Morris water maze. Here, we show for the first time that mice infected with Nippostrongylus brasiliensis were able to learn the Morris water maze task, but demonstrated impaired reference memory. Anxiety measured by thigmotaxis in the maze, did not play a role for the observed cognitive impairment. Of further interest, an increase in the number of hippocampal macrophages and microglia with training and/or infection suggested a significant role of these cell types during spatial learning. Together, these experimental mouse studies suggest that helminth infections do have an impact on cognition. Further experimental animal studies on cognition and infection might open new approaches for a better understanding and impact of pathogen infections.