Nippostrongylus Brasiliensis Research Articles

The role of ILC2 in hookworm infection.

Hookworm is a major public health concern, yet still relatively little is known about the immunological responses involved in human infection. Animal studies are mainly confined to using the natural rodent helminth Nippostrongylus brasiliensis as this has been proposed as the most accurate model of hookworm infection in the mouse, with both its life cycle and the immune responses it invokes having been extremely well characterized. In this review, we examine the roles that type 2 innate lymphoid cells (ILC2s) play in immunity and host tolerance to hookworm infection, particularly N.brasiliensis. This includes their role in the initiation and regulation of immune responses, as well as in the resolution and limitation of tissue damage required after an infection with a large organism, such as a helminth.

Research Note. Occurrence of gastrointestinal helminths in commensal rodents from Tabasco, Mexico

Summary The aim of this study was to determine the prevalence and species composition of helminths in commensal rodents captured inside private residences in the city of Villahermosa in Tabasco, Mexico. Trapping was performed at each house for three consecutive nights from October to December 2015. Fifty commensal rodents were captured: 23 Rattus norvegicus, 16 Mus musculus and 11 Rattus rattus. Rodents were transported alive to the laboratory and held in cages until they defecated. Feces were analyzed for helminth eggs using the Sheather’s flotation technique. The overall prevalence of helminths in rodents was 60 %: R. norvegicus was more likely to be parasitized (87.0 %) than R. rattus (63.6 %) and M. musculus (18.8 %). Eggs from at least 13 species of helminths were identified: Hymenolepis diminuta, Rodentolepis nana, Moniliformis moniliformis, Heligmosomoides polygyrus, Heterakis spumosa, Mastophorus muris, Nippostrongylus brasiliensis, Strongyloides ratti, Syphacia obvelata, Syphacia muris, Toxocara sp., Trichosomoides crassicauda, and Trichuris muris. This is the first study to report the presence of H. polygyrus, S. ratti and T. crassicauda in commensal rodents in Mexico. In conclusion, our results suggest that helminths commonly infect commensal rodents in Villahermosa and therefore rodents present a health risk to inhabitants in this region.

PD-1 regulates KLRG1+ group 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC-2s) regulate immune responses to pathogens and maintain tissue homeostasis in response to cytokines. Positive regulation of ILC-2s through ICOS has been recently elucidated. We demonstrate here that PD-1 is an important negative regulator of KLRG1+ ILC-2 function in both mice and humans. Increase in KLRG1+ ILC-2 cell numbers was attributed to an intrinsic defect in PD-1 signaling, which resulted in enhanced STAT5 activation. During Nippostrongylus brasiliensis infection, a significant expansion of KLRG1+ ILC-2 subsets occurred in Pdcd1-/- mice and, upon adoptive transfer, Pdcd1-/- KLRG1+ ILC-2s significantly reduced worm burden. Furthermore, blocking PD-1 with an antibody increased KLRG1+ ILC-2 cell number and reduced disease burden. Therefore, PD-1 is required for maintaining the number, and hence function, of KLRG1+ ILC-2s.

Helminth-induced B lymphocytes mediate control of emphysema through production of RELMa

Abstract The intestinal nematode parasite, Nippostrongylus brasiliensis (Nb), can cause emphysema and the mechanisms remain poorly defined. Here we show that emphysema develops as early as 7 days after Nb inoculation and is associated with increased gene expression of Il17a, neutrophil elastase (Ela2), and macrophage matrix metalloproteinase 12 (Mmp12) in the lung. Transfer of Nb-primed neutrophils or macrophages to naïve recipients results in emphysema. Nb infection of Il17a−/−mice results in a less severe emphysematous pathology, decreased neutrophil influx to the lung, and lower gene expression of Ela2. Surprisingly, Nb infection in B cell deficient Jh−/− mice results in a more severe emphysema when compared to WT mice, and is associated with higher numbers of neutrophils and macrophages, as well as increased expression of Il17a, Ela2 and Mmp12 in the lung. Transfer of B lymphocytes from WT mice or Il10−/− mice, but not from Il4Rα−/− mice, or Relmα−/− mice to recipient Jh−/−mice can restore control of emphysema, decrease influx of neutrophils, macrophages, and downregulate IL-17A. B cells from lung tissue of Nb-primed WT mice, but not Il4Rα−/− mice express high levels of Relmα. Co-culture of Nb-primed WT B cells but not Relmα−/− B cells with Nb-primed neutrophils or Nb-primed macrophages decreased ELA-2 and MMP-12 activity, respectively. Taken together, these data suggest that IL-17A promotes emphysema through recruitment and activation of protease secreting neutrophils and macrophages in the lung. IL4R-dependent B regulatory cells control emphysema through two independent mechanisms, downregulation of IL-17A and suppression of myeloid cell protease production, both of which require secretion of RELMα.

EFhd2/ Swiprosin-1 regulates the cytoskeleton of B lymphocytes and impacts germinal center reaction

Abstract The germinal center (GC) reaction is a decisive checkpoint for efficient humoral immunity. Most GC associated processes, e.g. cell migration and cell adhesion, are strongly dependent on cytoskeletal dynamics and B cell receptor (BCR) signaling. However, the molecular connection between downstream BCR molecules and cytoskeletal proteins is not yet fully elucidated. The adaptor protein EFhd2/ Swiprosin-1 co-localizes with the BCR at the plasma membrane. It controls F-Actin bundling as well as Actin remodeling through Cofilin activity and is involved in spreading, lamellipodia formation and migration in non-lymphoid cells. We showed that constitutively EFhd2 deficient mice display enhanced GC responses upon challenge with the helminth Nippostrongylus brasiliensis in a B cell intrinsic manner in mixed bone marrow chimeras. Moreover, B cell specific deletion of EFhd2 leads to enhanced GC B cell numbers already in steady state. We therefore hypothesize that EFhd2 controls the GC reaction by regulating cytoskeletal processes. In vitro transwell migration assays and in vivo studies using two-photon microscopy revealed decreased directed chemotaxis but increased random chemokinesis of EFhd2−/− B cells. Moreover, activated B cells spread faster in the absence of EFhd2 on anti-BCR antibodies which correlates with accelerated Cofilin activity. Finally, also BCR capping was increased in activated EFhd2 deficient B cells. Taken together, we suggest that EFhd2 controls Actin dynamics through Cofilin in B cells, thereby, controlling chemotaxis of B cells and BCR mediated B cell spreading. Hence, EFhd2 links proper coordination of the B cell cytoskeleton with the GC checkpoint and impacts TH2 response.

B1 cell IgE impedes B2 cell IgE-mediated parasite expulsion

Abstract Helminth infection is known for generating large amounts of poly-specific IgE. Previously we reported that innate-like B1 cells are responsible for poly-specific IgE production during infection with the nematode parasites Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. We showed that B1 cells make large amounts of IgE. In vitro analysis of factors critical for B1 cell immunoglobulin class switch recombination to IgE demonstrated a requirement for anti-CD40 and IL-4 that further enhanced IgE production when IL-5 was added to culture or when the B1 source was helminth infected mice. We additionally reported that there was also a novel IL-25-induced upregulation of IgE in B1 cells isolated from helminth infected mice. Here, we report that in T cell reconstituted RAG1−/− mice further reconstituted with or without B1 cells and/or B2 cells and followed by infection with N. brasiliensis only B2-reconstituted mice had significantly reduced fecal egg production that was IgE dependent. Parasite clearance was impeded by reconstitution with B1 cells and was also IgE dependent. Helminth-induced B1 cell IgE was not specific for N. brasiliensis because it blocked mast cell degranulation to N. brasiliensis excretory secretory antigens in infected mice. The data supports a hypothesis that B1 cell IgE secretion has evolved as a protective response used by the helminth. In searching for a human B cell equivalent, we additionally identified a novel population of human tonsilar B cells that respond to IL-25 stimulation with increased proliferation and IgE production. We hypothesize that these cells may be important in generation of human poly-specific IgE.

Downregulation of E Protein Activity Augments an ILC2 Differentiation Program in the Thymus.

Innate lymphoid cells (ILCs) are important regulators in various immune responses. The current paradigm states that all newly made ILCs originate from common lymphoid progenitors in the bone marrow. Id2, an inhibitor of E protein transcription factors, is indispensable for ILC differentiation. Unexpectedly, we found that ectopically expressing Id1 or deleting two E protein genes in the thymus drastically increased ILC2 counts in the thymus and other organs where ILC2 normally reside. Further evidence suggests a thymic origin of these mutant ILC2s. The mutant mice exhibit augmented spontaneous infiltration of eosinophils and heightened responses to papain in the lung and increased ability to expulse the helminth parasite, Nippostrongylus brasiliensis These results prompt the questions of whether the thymus naturally has the capacity to produce ILC2s and whether E proteins restrain such a potential. The abundance of ILC2s in Id1 transgenic mice also offers a unique opportunity for testing the biological functions of ILC2s.

Annotated mitochondrial genome with Nanopore R9 signal for Nippostrongylus brasiliensis

Nippostrongylus brasiliensis, a nematode parasite of rodents, has a parasitic life cycle that is an extremely useful model for the study of human hookworm infection, particularly in regards to the induced immune response. The current reference genome for this parasite is highly fragmented with minimal annotation, but new advances in long-read sequencing suggest that a more complete and annotated assembly should be an achievable goal. We de-novo assembled a single contig mitochondrial genome from N. brasiliensis using MinION R9 nanopore data. The assembly was error-corrected using existing Illumina HiSeq reads, and annotated in full (i.e. gene boundary definitions without substantial gaps) by comparing with annotated genomes from similar parasite relatives. The mitochondrial genome has also been annotated with a preliminary electrical consensus sequence, using raw signal data generated from a Nanopore R9 flow cell.

IGF1 Shapes Macrophage Activation in Response to Immunometabolic Challenge

In concert with their phagocytic activity, macrophages are thought to regulate the host immunometabolic responses primarily via their ability to produce specific cytokines and metabolites. Here, we show that IL-4-differentiated, M2-like macrophages secrete IGF1, a hormone previously thought to be exclusively produced from liver. Ablation of IGF1 receptors from myeloid cells reduced phagocytosis, increased macrophages in adipose tissue, elevated adiposity, lowered energy expenditure, and led to insulin resistance in mice fed a high-fat diet. The investigation of adipose macrophage phenotype in obese myeloid IGF1R knockout (MIKO) mice revealed a reduction in transcripts associated with M2-like macrophage activation. Furthermore, the MIKO mice infected with helminth Nippostrongylus brasiliensis displayed delayed resolution from infection with normal insulin sensitivity. Surprisingly, cold challenge did not trigger an overt M2-like state and failed to induce tyrosine hydroxylase expression in adipose tissue macrophages of control or MIKO mice. These results show that IGF1 signaling shapes the macrophage-activation phenotype.

Annotated mitochondrial genome with Nanopore R9 signal for Nippostrongylus brasiliensis.

Nippostrongylus brasiliensis, a nematode parasite of rodents, has a parasitic life cycle that is an extremely useful model for the study of human hookworm infection, particularly in regards to the induced immune response. The current reference genome for this parasite is highly fragmented with minimal annotation, but new advances in long-read sequencing suggest that a more complete and annotated assembly should be an achievable goal. We de-novoassembled a single contig mitochondrial genome from N. brasiliensisusing MinION R9 nanopore data. The assembly was error-corrected using existing Illumina HiSeq reads, and annotated in full (i.e. gene boundary definitions without substantial gaps) by comparing with annotated genomes from similar parasite relatives. The mitochondrial genome has also been annotated with a preliminary electrical consensus sequence, using raw signal data generated from a Nanopore R9 flow cell.

Th9 Cells and Parasitic Inflammation: Use of Nippostrongylus and Schistosoma Models.

Th9 cells are a new subpopulation of CD4+ T helper cells, characterized by the expression of IL-9 that have been involved in type 2 immune responses, antitumor responses and autoimmune diseases. Here, we describe two different parasitic models frequently maintained in the laboratory where Th9 cells or IL-9 (the cytokine produced by Th9 cells) has been shown to play critical roles in pathogen clearance and immune response activation: the nematode Nippostrongylus brasiliensis and the trematode Schistosoma mansoni.

Th2 responses are primed by skin dendritic cells with distinct transcriptional profiles.

The dendritic cell signals required for the in vivo priming of IL-4-producing T cells are unknown. We used RNA sequencing to characterize DCs from skin LN of mice exposed to two different Th2 stimuli: the helminth parasite Nippostrongylus brasiliensis (Nb) and the contact sensitizer dibutyl phthalate (DBP)-FITC. Both Nb and DBP-FITC induced extensive transcriptional changes that involved multiple DC subsets. Surprisingly, these transcriptional changes were highly distinct in the two models, with only a small number of genes being similarly regulated in both conditions. Pathway analysis of expressed genes identified no shared pathways between Nb and DBP-FITC, but revealed a type-I IFN (IFN-I) signature unique to DCs from Nb-primed mice. Blocking the IFN-I receptor at the time of Nb treatment had little effect on DC migration and antigen transport to the LN, but inhibited the up-regulation of IFN-I-induced markers on DCs and effectively blunted Th2 development. In contrast, the response to DBP-FITC was not affected by IFN-I receptor blockade, a finding consistent with the known dependence of this response on the innate cytokine TSLP. Thus, the priming of Th2 responses is associated with distinct transcriptional signatures in DCs in vivo, reflecting the diverse environments in which Th2 immune responses are initiated.

Molecular Characterization of Nippostrongylus brasiliensis (Nematoda: Heligmosomatidae) from Mus musculus in India

Mus musculus (Rodentia: Muridae) has generally been infected with a rodent hookworm Nippostrongylus brasiliensis. In this report, we present morphological and molecular identification of N. brasiliensis by light and scanning electron microscopy and PCR amplification of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the protein sequences encoded by cox1 gene, respectively. Despite the use of N. brasiliensis in many biochemistry studies from India, their taxonomic identification was not fully understood, especially at the species level, and no molecular data is available in GenBank from India. Sequence analysis of cox1 gene in this study revealed that the present specimen showed close identity with the same species available in GenBank, confirming that the species is N. brasiliensis. This study represents the first record of molecular identification of N. brasiliensis from India and the protein structure to better understand the comparative phylogenetic characteristics.

Modulation of the Immune Response by Nematode Secreted Acetylcholinesterase Revealed by Heterologous Expression in Trypanosoma musculi

Nematode parasites secrete molecules which regulate the mammalian immune system, but their genetic intractability is a major impediment to identifying and characterising the biological effects of these molecules. We describe here a novel system for heterologous expression of helminth secreted proteins in the natural parasite of mice, Trypanosoma musculi, which can be used to analyse putative immunomodulatory functions. Trypanosomes were engineered to express a secreted acetylcholinesterase from Nippostrongylus brasiliensis. Infection of mice with transgenic parasites expressing acetylcholinesterase resulted in truncated infection, with trypanosomes cleared early from the circulation. Analysis of cellular phenotypes indicated that exposure to acetylcholinesterase in vivo promoted classical activation of macrophages (M1), with elevated production of nitric oxide and lowered arginase activity. This most likely occurred due to the altered cytokine environment, as splenocytes from mice infected with T. musculi expressing acetylcholinesterase showed enhanced production of IFNγ and TNFα, with diminished IL-4, IL-13 and IL-5. These results suggest that one of the functions of nematode secreted acetylcholinesterase may be to alter the cytokine environment in order to inhibit development of M2 macrophages which are deleterious to parasite survival. Transgenic T. musculi represents a valuable new vehicle to screen for novel immunoregulatory proteins by extracellular delivery in vivo to the murine host.

Peeking into immunoregulatory effects of phototherapy.

Peeking into immunoregulatory effects of phototherapy.

Th2 and eosinophil responses suppress inflammatory arthritis.

Th2–eosinophil immune responses are well known for mediating host defence against helminths. Herein we describe a function of Th2–eosinophil responses in counteracting the development of arthritis. In two independent models of arthritis, Nippostrongylus brasiliensis infection leads to Th2 and eosinophil accumulation in the joints associated with robust inhibition of arthritis and protection from bone loss. Mechanistically, this protective effect is dependent on IL-4/IL-13-induced STAT6 pathway. Furthermore, we show that eosinophils play a central role in the modulation of arthritis probably through the increase of anti-inflammatory macrophages into arthritic joints. The presence of these pathways in human disease is confirmed by detection of GATA3-positive cells and eosinophils in the joints of rheumatoid arthritis patients. Taken together, these results demonstrate that eosinophils and helminth-induced activation of the Th2 pathway axis effectively mitigate the course of inflammatory arthritis.

Depletion of major pathogenic cells in asthma by targeting CRTh2

Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and Nippostrongylus brasiliensis infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2+ basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.

Examining the function of chemokine receptor deficient-monocytes during infection with Nippostrongylus brasiliensis

Abstract Nippostrongylus brasiliensis (Nb) is a murine helminth that acutely infects the lungs and the small intestine, and induces a strong Th2 immune response, which includes contributions from innate immune cells such as monocytes. CCR2, the receptor for MCP1, which is expressed primarily on monocytes, was predictably important for monocyte recruitment during Nb infection. However, it was also shown to be necessary to control hemorrhaging as CCR2−/− mice had more blood in the bronchoalveolar lavage fluid compared to wild-type (WT) mice. In addition, CCR2−/− mice produced higher egg burden and more viable worms based on parasite ATP assays. Signaling through CX3CR1, another receptor expressed on monocytes provoked the opposite phenotype. CX3CR1 was not crucial for cellular recruitment after infection, but Nb from CX3CR1−/− mice produced less eggs and were less viable than Nb from WT mice. To investigate potential mechanisms by which monocytes influence worm viability, infective stage Nb (L3) were co-cultured with sorted Ly6c+ monocytes from CX3CR1 WT or knockout (ko) mice. After 5 days in culture, Nb incubated with ko Ly6c+ cells were less motile than Nb cultured with WT Ly6c+ cells. Taken together, these data indicate that CCR2-dependent infiltrating monocytes control inflammation and promote worm killing, while CX3CR1 signaling in monocytes promotes worm viability potentially by influencing worm motility.

IL-4R signaling in B cells mediates control of IL-17A dependent emphysema triggered by N. brasiliensis infection.

Abstract The intestinal nematode parasite, Nippostrongylus brasiliensis (Nb), transiently resides in the lung and causes lung damage, which is then rapidly repaired. However, the repaired lung eventually develops emphysema and the mechanisms contributing to this chronic lung pathology remain poorly defined. Here we show that emphysema, as measured by mean linear intercept measurements (Lm) of alveolar spaces, develops as early as 7 days after Nb inoculation and is associated with increased gene expression of Il17A, neutrophil elastase (ELA), and macrophage matrix metalloproteinase 12 (Mmp12) in the lung. Transfer of Nb-primed neutrophils or macrophages to naïve recipients results in emphysema, and these cells expressed high level of ELA and Mmp12, respectively. Neutralization of IL-17A using anti-IL-17A antibody curbed development of emphysema, and decreased influx of neutrophils in the lung and gene expression of Il17A. Parasite infection in B cell deficient Jh−/− mice results in a more severe emphysema when compared to wild-type mice or FcRγ−/− mice, and is associated with higher numbers of neutrophils and macrophages, as well as increased expression of Il17a, ELA2 and Mmp12 in the lung. Transfer of B lymphocytes from wild-type mice or IL-10−/− mice, but not from IL4Rα−/− mice, to recipient Jh−/− mice can restore control of emphysema and downregulate IL-17A. B cells from lung tissue of Nb-primed wild type but not IL4Rα−/− mice expressed high level of Relmα. Taken together, these data suggest that IL-17A triggers infiltration of neutrophils and macrophages that contribute to chronic emphysema, which is controlled by IL-4R dependent B regulatory cells expressing high levels of Relmα.

Helminth-induced B1 IgE is protective against allergic disease.

Abstract Helminth infection is well known for its ability to generate large amounts of non-specific IgE. This hinders the parasite-specific IgE thereby reducing mast cell (MC) degranulation and anti-helminth immunity. This study shows that the innate-like B1 cells are responsible for the substantial amounts of IgE induced during the Th2 helminth infection, Nippostrongylus brasiliensis (Nb). We utilize a mouse model with defects in hematopoiesis that results in loss of B2 cells, but retains normal B1 development, the ADAM10 transgenic mouse. These mice have equivalent IgE production in Nb when compared to wild type (WT) controls that is T cell dependent. Utilizing the NP-KLH system, we demonstrate that these mice make no antigen-specific IgG1, either high or total affinity. They additionally make no increased overall antibody when boosted with the same antigen. In WT mice, we show a population of B1 plasma cells (CD11b+CD23−B220intCD138+) in vivo, that express secreted IgE message during Nb infection. We additionally show that B1 cells (CD11b+CD23−B220int) sorted from naïve and Nb infected mice make large amounts of IgE in in vitro culture with IL-4, anti-CD40, and IL-5 by ELISA and secreted IgE message by RT-PCR. These B1 cells become CD138+ after 5 days of culture. Finally and most importantly, we show that B1 IgE is first unable to induce MC degranulation in an active cutaneous anaphylaxis model and second, it can block MC degranulation in a model of passive cutaneous anaphylaxis. Overall, these data represents a protective response that has evolved for the helminth, where non-specific B1 IgE blocks MC degranulation and thereby protects the helminth from damage. Excitingly, this could be harnessed as a potential protection against allergic disease.