ABSTRACT
Intestinal helminth infection triggers a type 2 immune response that promotes a 'weep-and sweep' response characterised by increased mucus secretion and intestinal hypermotility, which function to dislodge the worm from its intestinal habitat. Recent studies have discovered that several other pathogens cause intestinal dysmotility through major alterations to the immune and enteric nervous systems (ENS), and their interactions, within the gastrointestinal tract. However, the involvement of these systems has not been investigated for helminth infections. Eosinophils represent a key cell type recruited by the type 2 immune response and alter intestinal motility under steady-state conditions. Our study aimed to investigate whether altered intestinal motility driven by the murine hookworm, Nippostrongylus brasiliensis, infection involves eosinophils and how the ENS and smooth muscles of the gut are impacted. Eosinophil deficiency did not influence helminth-induced intestinal hypermotility and hypermotility did not involve gross structural or functional changes to the ENS. Hypermotility was instead associated with a dramatic increase in smooth muscle thickness and contractility, an observation that extended to another rodent nematode, Heligmosomoides polygyrus. In summary our data indicate that, in contrast to other pathogens, helminth-induced intestinal hypermotility is driven by largely by myogenic, rather than neurogenic, alterations with such changes occurring independently of eosinophils. (<300 words).
Subject(s)
Enteric Nervous System , Eosinophils , Gastrointestinal Motility , Muscle, Smooth , Nippostrongylus , Animals , Mice , Eosinophils/immunology , Muscle, Smooth/parasitology , Enteric Nervous System/parasitology , Enteric Nervous System/immunology , Gastrointestinal Motility/physiology , Nematospiroides dubius/physiology , Nematospiroides dubius/immunology , Strongylida Infections/immunology , Strongylida Infections/parasitology , Intestinal Diseases, Parasitic/immunology , Intestinal Diseases, Parasitic/parasitology , Helminthiasis/immunology , Helminthiasis/parasitology , Neurons/parasitology , Neurons/metabolism , Mice, Inbred C57BLABSTRACT
Increased permeability of the intestinal epithelial layer is linked to the pathogenesis and perpetuation of a wide range of intestinal and extra-intestinal diseases. Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed as a treatment for many of the same diseases. Helminths induce immunoregulatory changes in their host which could decrease epithelial permeability, which is highlighted as a potential mechanism through which helminths treat disease. Despite this, the influence of a chronic helminth infection on epithelial permeability remains unclear. This study uses the chronically infecting intestinal helminth Heligmosomoides polygyrus to reveal alterations in the expression of intestinal tight junction proteins and epithelial permeability during the infection course. In the acute infection phase (1 week postinfection), an increase in intestinal epithelial permeability is observed. Consistent with this finding, jejunal claudin-2 is upregulated and tricellulin is downregulated. By contrast, in the chronic infection phase (6 weeks postinfection), colonic claudin-1 is upregulated and epithelial permeability decreases. Importantly, this study also investigates changes in epithelial permeability in a small human cohort experimentally challenged with the human hookworm, Necator americanus. It demonstrates a trend toward small intestinal permeability increasing in the acute infection phase (8 weeks postinfection), and colonic and whole gut permeability decreasing in the chronic infection phase (24 weeks postinfection), suggesting a conserved epithelial response between humans and mice. In summary, our findings demonstrate dynamic changes in epithelial permeability during a chronic helminth infection and provide another plausible mechanism by which chronic helminth infections could be utilized to treat disease.
Subject(s)
Intestinal Mucosa , Permeability , Animals , Humans , Intestinal Mucosa/parasitology , Intestinal Mucosa/metabolism , Intestinal Mucosa/immunology , Chronic Disease , Nematospiroides dubius/immunology , Mice , Necator americanus , Intestinal Diseases, Parasitic/immunology , Tight Junctions/metabolism , Tight Junction Proteins/metabolism , Intestine, Small/parasitology , Intestine, Small/immunology , Female , Mice, Inbred C57BL , Male , Helminthiasis/immunology , Helminthiasis/parasitology , Necatoriasis/immunology , MARVEL Domain Containing 2 Protein/metabolismABSTRACT
BACKGROUND: Human hookworm has been proposed as a treatment for ulcerative colitis (UC). This pilot study assessed the feasibility of a full-scale randomized control trial examining hookworm to maintain clinical remission in patients with UC. METHODS: Twenty patients with UC in disease remission (Simple Clinical Colitis Activity Index [SCCAI] ≤4 and fecal calprotectin (fCal) <100 ug/g) and only on 5-aminosalicylate received 30 hookworm larvae or placebo. Participants stopped 5-aminosalicylate after 12 weeks. Participants were monitored for up to 52 weeks and exited the study if they had a UC flare (SCCAI ≥5 and fCal ≥200 µg/g). The primary outcome was difference in rates of clinical remission at week 52. Differences were assessed for quality of life (QoL) and feasibility aspects including recruitment, safety, effectiveness of blinding, and viability of the hookworm infection. RESULTS: At 52 weeks, 4 of 10 (40%) participants in the hookworm group and 5 of 10 (50%) participants in the placebo group had maintained clinical remission (odds ratio, 0.67; 95% CI, 0.11-3.92). Median time to flare in the hookworm group was 231 days (interquartile range [IQR], 98-365) and 259 days for placebo (IQR, 132-365). Blinding was quite successful in the placebo group (Bang's blinding index 0.22; 95% CI, -0.21 to 1) but less successful in the hookworm group (0.70; 95% CI, 0.37-1.0). Almost all participants in the hookworm group had detectable eggs in their faeces (90%; 95% CI, 0.60-0.98), and all participants in this group developed eosinophilia (peak eosinophilia 4.35â ×â 10^9/L; IQR, 2.80-6.68). Adverse events experienced were generally mild, and there was no significant difference in QoL. CONCLUSIONS: A full-scale randomized control trial examining hookworm therapy as a maintenance treatment in patients with UC appears feasible.
This pilot study has shown a full-scale RCT examining hookworm therapy as maintenance therapy in patients with ulcerative colitis is feasible, safe, and will be well-tolerated.
ABSTRACT
Recent advances in the field of host immunity against parasitic nematodes have revealed the importance of macrophages in trapping tissue migratory larvae. Protective immune mechanisms against the rodent hookworm Nippostrongylus brasiliensis (Nb) are mediated, at least in part, by IL-4-activated macrophages that bind and trap larvae in the lung. However, it is still not clear how host macrophages recognize the parasite. An in vitro co-culture system of bone marrow-derived macrophages and Nb infective larvae was utilized to screen for the possible ligand-receptor pair involved in macrophage attack of larvae. Competitive binding assays revealed an important role for ß-glucan recognition in the process. We further identified a role for CD11b and the non-classical pattern recognition receptor ephrin-A2 (EphA2), but not the highly expressed ß-glucan dectin-1 receptor, in this process of recognition. This work raises the possibility that parasitic nematodes synthesize ß-glucans and it identifies CD11b and ephrin-A2 as important pattern recognition receptors involved in the host recognition of these evolutionary old pathogens. To our knowledge, this is the first time that EphA2 has been implicated in immune responses to a helminth.
Subject(s)
Interleukin-4 , Lectins, C-Type , Ancylostomatoidea , Animals , Interleukin-4/metabolism , Larva , Lectins, C-Type/metabolism , Macrophages/metabolism , Receptors, ImmunologicABSTRACT
BACKGROUND: The type 2 cytokines IL-4 and IL-13 promote not only atopic dermatitis (AD) but also the resolution of inflammation. How type 2 cytokines participate in the resolution of AD is poorly known. OBJECTIVE: Our aim was to determine the mechanisms and cell types governing skin inflammation, barrier dysfunction, and resolution of inflammation in a model of AD. METHODS: Mice that exhibit expression of IL-4, IL-13, and MCPT8 or that could be depleted of basophils or eosinophils, be deficient in IL-4 or MHC class II molecules, or have basophils lacking macrophage colony-stimulating factor (M-CSF) were treated with calcipotriol (MC903) as an acute model of AD. Kinetics of the disease; keratinocyte differentiation; and leukocyte accumulation, phenotype, function, and cytokine production were measured by transepidermal water loss, histopathology, molecular biology, or unbiased analysis of spectral flow cytometry. RESULTS: In this model of AD, basophils were activated systemically and were the initial and main source of IL-4 in the skin. Basophils and IL-4 promoted epidermal hyperplasia and skin barrier dysfunction by acting on keratinocyte differentiation during inflammation. Basophils, IL-4, and basophil-derived M-CSF inhibited the accumulation of proinflammatory cells in the skin while promoting the expansion and function of proresolution M2-like macrophages and the expression of probarrier genes. Basophils kept their proresolution properties during AD resolution. CONCLUSION: Basophils can display both beneficial and detrimental type 2 functions simultaneously during atopic inflammation.
Subject(s)
Basophils/immunology , Dermatitis, Atopic/immunology , Skin/immunology , Animals , Calcitriol/analogs & derivatives , Cell Differentiation , Cytokines/genetics , Cytokines/immunology , Dermatitis, Atopic/chemically induced , Dermatitis, Atopic/genetics , Dermatitis, Atopic/pathology , Diphtheria Toxin , Edema/chemically induced , Edema/immunology , Eosinophils/immunology , Female , Gene Expression , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Hyperplasia/immunology , Keratinocytes/cytology , Male , Mice, Inbred C57BL , Mice, Transgenic , Skin/pathologyABSTRACT
Mature basophils play critical inflammatory roles during helminthic, autoimmune, and allergic diseases through their secretion of histamine and the type 2 cytokines interleukin 4 (IL-4) and IL-13. Basophils are activated typically by allergen-mediated IgE cross-linking but also by endogenous "innate" factors. The aim of this study was to identify the innate stimuli (cytokines, chemokines, growth factors, hormones, neuropeptides, metabolites, and bacterial products) and signaling pathways inducing primary basophil activation. Basophils from naïve mice or helminth-infected mice were cultured with up to 96 distinct stimuli and their influence on basophil survival, activation, degranulation, and IL-4 or IL-13 expression were investigated. Activated basophils show a heterogeneous phenotype and segregate into distinct subsets expressing IL-4, IL-13, activation, or degranulation markers. We find that several innate stimuli including epithelial derived inflammatory cytokines (IL-33, IL-18, TSLP, and GM-CSF), growth factors (IL-3, IL-7, TGFß, and VEGF), eicosanoids, metabolites, TLR ligands, and type I IFN exert significant direct effects on basophils. Basophil activation mediated by distinct upstream signaling pathways is always sensitive to Syk and IκB kinases-specific inhibitors but not necessarily to NFAT, STAT5, adenylate cyclase, or c-fos/AP-1 inhibitors. Thus, basophils are activated by very diverse mediators, but their activation seem controlled by a core checkpoint involving Syk and IκB kinases.
Subject(s)
Basophils/immunology , Basophils/metabolism , I-kappa B Kinase/metabolism , Immunity, Innate , Signal Transduction , Syk Kinase/metabolism , Animals , Basophils/drug effects , Biomarkers , Cell Degranulation , Cytokines/metabolism , Gene Expression , Hormones , Immunity, Innate/drug effects , Inflammation Mediators/metabolism , Mice , Protein Kinase Inhibitors , Signal Transduction/drug effectsABSTRACT
Hookworms cause a major neglected tropical disease, occurring after larvae penetrate the host skin. Neutrophils are phagocytes that kill large pathogens by releasing neutrophil extracellular traps (NETs), but whether they target hookworms during skin infection is unknown. Using a murine hookworm, Nippostrongylus brasiliensis, we observed neutrophils being rapidly recruited and deploying NETs around skin-penetrating larvae. Neutrophils depletion or NET inhibition altered larvae behavior and enhanced the number of adult worms following murine infection. Nevertheless, larvae were able to mitigate the effect of NETs by secreting a deoxyribonuclease (Nb-DNase II) to degrade the DNA backbone. Critically, neutrophils were able to kill larvae in vitro, which was enhanced by neutralizing Nb-DNase II. Homologs of Nb-DNase II are present in other nematodes, including the human hookworm, Necator americanus, which also evaded NETs in vitro. These findings highlight the importance of neutrophils in hookworm infection and a potential conserved mechanism of immune evasion.
Subject(s)
Ancylostomatoidea/immunology , Endodeoxyribonucleases/biosynthesis , Extracellular Traps/metabolism , Immune Evasion , Animals , Host-Parasite Interactions , Mice , Neutrophils/metabolism , Nippostrongylus/immunology , Strongylida Infections/immunologyABSTRACT
Basophils are granulocytes involved in parasite immunity and allergic diseases, known for their potent secretion of type 2 cytokines. Identifying their functions has proven to be controversial due to their relative rarity and their complex lineage phenotype. Here, we show that the expression of basophils lineage markers CD200R3 and FcεRIα is highly variable in inflammatory settings and hinders basophils identification by flow cytometry across multiple disease states or tissues. Fluorophore-conjugated antibody staining of these lineage markers strongly activates basophil type 2 cytokine expression, and represents a potential bias for coculture or in vivo transfer experiments. The Basoph8 is a mouse model where basophils specifically express a strong fluorescent reporter and the Cre recombinase. Basophils can be identified and FACS sorted unambiguously by their expression of the enhanced yellow fluorescent protein (eYFP) in these mice. We show that the expression of the eYFP is robust in vivo during inflammation, and in vitro on living basophils for at least 72 h, including during the induction of anaphylactoid degranulation. We bred and characterized the Basoph8xiDTR mice, in which basophils specifically express eYFP and the simian diphtheria toxin receptor (DTR). This model enables basophils conditional depletion relatively specifically ex vivo and in vivo during allergic inflammation and their detection as eYFP+ cells. In conclusion, we report underappreciated benefits of the commercially available Basoph8 mice to study basophils function.
Subject(s)
Basophils/immunology , Hypersensitivity/immunology , Inflammation/immunology , Animals , Mice, Inbred C57BL , Skin/immunologyABSTRACT
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.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Coinfection , Eosinophils/immunology , Interleukin-5/metabolism , Lung/immunology , Nematospiroides dubius/physiology , Nippostrongylus/physiology , Strongylida Infections/immunology , Trichuriasis/immunology , Trichuris/physiology , Animals , Antigens, Helminth/immunology , Cell Movement , Cells, Cultured , Cytotoxicity, Immunologic , Female , Host-Parasite Interactions , Immunity , Interleukin-33/metabolism , Lung/parasitology , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
As part of on-going efforts to control hookworm infection, the "human hookworm vaccine initiative" has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm.
Subject(s)
Antibodies, Helminth/pharmacology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Erythrocytes/drug effects , Hookworm Infections/prevention & control , Necator americanus/enzymology , Nippostrongylus/growth & development , Strongylida Infections/prevention & control , Ancylostomatoidea/drug effects , Ancylostomatoidea/growth & development , Animals , Antigens, Helminth/immunology , Aspartic Acid Endopeptidases/immunology , Erythrocytes/parasitology , Female , Hookworm Infections/parasitology , Life Cycle Stages , Male , Mice , Mice, Inbred C57BL , Nippostrongylus/drug effects , Strongylida Infections/parasitologyABSTRACT
BACKGROUND: Eukaryotic genome assembly remains a challenge in part due to the prevalence of complex DNA repeats. This is a particularly acute problem for holocentric nematodes because of the large number of satellite DNA sequences found throughout their genomes. These have been recalcitrant to most genome sequencing methods. At the same time, many nematodes are parasites and some represent a serious threat to human health. There is a pressing need for better molecular characterization of animal and plant parasitic nematodes. The advent of long-read DNA sequencing methods offers the promise of resolving complex genomes. RESULTS: Using Nippostrongylus brasiliensis as a test case, applying improved base-calling algorithms and assembly methods, we demonstrate the feasibility of de novo genome assembly matching current community standards using only MinION long reads. In doing so, we uncovered an unexpected diversity of very long and complex DNA sequences repeated throughout the N. brasiliensis genome, including massive tandem repeats of tRNA genes. CONCLUSION: Base-calling and assembly methods have improved sufficiently that de novo genome assembly of large complex genomes is possible using only long reads. The method has the added advantage of preserving haplotypic variants and so has the potential to be used in population analyses.
Subject(s)
Genome, Helminth/genetics , High-Throughput Nucleotide Sequencing/methods , Nippostrongylus/genetics , Sequence Analysis, DNA/methods , Animals , Base Sequence/genetics , Female , Nippostrongylus/isolation & purification , Rats , Rats, Inbred Lew , Rats, Sprague-DawleyABSTRACT
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.
ABSTRACT
Polymorphisms in genes involved in IL-4 responses segregate with allergic disease risk and correlate with IgE levels in humans, and IL-4 promotes IgE and IgG1 Ab production against allergens in mice. We report that mice with only one intact Il4 gene copy are significantly impaired in their ability to make specific IgE responses against allergens, whereas IgG1 responses to allergens remain unaffected. Il4-hemizygosity also resulted in a modest but detectable drop in IL-4 production by CD4+ T cells isolated from lymph nodes and prevented IgE-dependent oral allergen-induced diarrhea. We conclude that a state of haploinsufficiency for the Il4 gene locus is specifically relevant for IL-4-dependent IgE responses to allergens with the amount of IL-4 produced in the hemizygous condition falling close to the threshold required for switching to IgE production. These results may be relevant for how polymorphisms in genes affecting IL-4 responses influence the risk of IgE-mediated allergic disease in humans.
Subject(s)
Allergens/immunology , Haploinsufficiency , Immunoglobulin E/immunology , Interleukin-4/genetics , Animals , CD4-Positive T-Lymphocytes/immunology , Hypersensitivity/immunology , Immunoglobulin E/biosynthesis , Immunoglobulin G/immunology , Interleukin-4/immunology , Mice , Pollen/immunology , Polymorphism, GeneticABSTRACT
Defining the immune mechanisms underlying protective immunity to helminth infection remains an important challenge. Here we report that lung CD4(+) T cells and Group 2 innate lymphoid cells (ILC2s) work in concert to block Nippostrongylus brasiliensis (Nb) development in the parenchyma within 48 h in mice. Immune-damaged larvae have a striking morphological defect that is dependent on the expansion of IL-13-producing ILC2 and CD4(+) T cells, and the activation of M2 macrophages. This T-cell requirement can be bypassed by administration of IL-2 or IL-33, resulting in expansion of IL-13-producing ILC2s and larval killing. Depletion of ILC2s inhibits larval killing in IL-2-treated mice. Our results broaden understanding of ILC2's role in immunity to helminths by demonstrating that they not only act as alarmin sensors, but can also be sustained by CD4(+) T cells, ensuring both the prompt activation and the maintenance of IL-13-dependent M2 macrophage immunity in the lung.
Subject(s)
CD4-Positive T-Lymphocytes/physiology , Host-Pathogen Interactions/immunology , Lung/immunology , Macrophages/physiology , Nippostrongylus/physiology , Animals , Interleukin-2/metabolism , Interleukin-33/metabolism , Mice, Inbred BALB C , Neutrophils/physiology , Rats, Inbred LewABSTRACT
Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.
Subject(s)
Antibodies, Helminth/immunology , Antigens, Helminth/immunology , Immunoglobulin G/immunology , Interleukin-4/immunology , Interleukins/immunology , Nematospiroides dubius/immunology , Strongylida Infections/immunology , Vaccination , Animals , Antibodies, Helminth/genetics , Humans , Immunoglobulin G/genetics , Interleukin-4/genetics , Interleukins/genetics , Larva/immunology , Mice , Mice, Knockout , Strongylida Infections/genetics , Strongylida Infections/prevention & controlABSTRACT
Parasitic helminth infections can be associated with lifelong morbidity such as immune-mediated organ failure. A better understanding of the host immune response to helminths could provide new avenues to promote parasite clearance and/or alleviate infection-associated morbidity. Murine resistin-like molecules (RELM) exhibit pleiotropic functions following helminth infection including modulating the host immune response; however, the relevance of human RELM proteins in helminth infection is unknown. To examine the function of human resistin (hResistin), we utilized transgenic mice expressing the human resistin gene (hRetnTg+). Following infection with the helminth Nippostrongylus brasiliensis (Nb), hResistin expression was significantly upregulated in infected tissue. Compared to control hRetnTg- mice, hRetnTg+ mice suffered from exacerbated Nb-induced inflammation characterized by weight loss and increased infiltration of inflammatory monocytes in the lung, along with elevated Nb egg burdens and delayed parasite expulsion. Genome-wide transcriptional profiling of the infected tissue revealed that hResistin promoted expression of proinflammatory cytokines and genes downstream of toll-like receptor signaling. Moreover, hResistin preferentially bound lung monocytes, and exogenous treatment of mice with recombinant hResistin promoted monocyte recruitment and proinflammatory cytokine expression. In human studies, increased serum resistin was associated with higher parasite load in individuals infected with soil-transmitted helminths or filarial nematode Wuchereria bancrofti, and was positively correlated with proinflammatory cytokines. Together, these studies identify human resistin as a detrimental factor induced by multiple helminth infections, where it promotes proinflammatory cytokines and impedes parasite clearance. Targeting the resistin/proinflammatory cytokine immune axis may provide new diagnostic or treatment strategies for helminth infection and associated immune-mediated pathology.
Subject(s)
Helminthiasis , Inflammation Mediators/metabolism , Macrophages/metabolism , Monocytes/metabolism , Parasitemia/genetics , Resistin/genetics , Animals , Cytokines/metabolism , Female , Helminthiasis/genetics , Helminthiasis/immunology , Helminthiasis/metabolism , Helminthiasis/parasitology , Humans , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Inflammation/parasitology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Monocytes/immunology , Nippostrongylus/immunology , Parasitemia/immunology , Parasitemia/metabolism , Rats , Rats, Sprague-Dawley , Resistin/metabolism , Strongylida Infections/genetics , Strongylida Infections/immunology , Strongylida Infections/metabolism , Strongylida Infections/parasitology , Up-Regulation/geneticsABSTRACT
Dendritic cells (DC) are critical for the initiation of immune responses; however, their role in priming IL-4-producing Th2 cells in vivo is not fully understood. We used a model of intradermal injection with fluorescent-labeled, nonviable larvae from the helminth parasite nonviable Nippostrongylus brasiliensis L3 larvae (Nb), a strong inducer of Th2 responses, together with IL-4-GFP reporter mice that enable a sensitive detection of IL-4 production to examine the contribution of DC to the priming of IL-4-producing CD4(+) T cells in vivo. We found that parasite material is taken up by two distinct DC populations in draining lymph nodes: a mostly CD11c(int)MHC class II (MHCII)(hi)CD11b(+)Ly6C(-) dermal DC population and a CD11c(hi)MHCII(int)CD11b(+)Ly6C(+) monocyte-derived DC population. After Nb treatment, these two DC populations appeared in the draining lymph nodes in comparable numbers and with similar kinetics; however, treatment with pertussis toxin blocked the migration of dermal DC and the priming of IL-4-producing T cells, but only partially affected monocyte-derived DC numbers. In line with this observation, transfer of OVA-loaded CD11c(int)MHCII(hi) DC from Nb-treated mice into naive hosts could sensitize OVA-specific CD4(+) T cells to IL-4 production, whereas transfer of CD11c(int)MHCII(hi) DC from naive mice, or CD11c(hi)MHCII(int) DC from Nb-treated or naive mice, induced CD4(+) T cell expansion but no IL-4 production. Phenotypic analysis of Nb-loaded CD11c(int)MHCII(hi) DC revealed expression of programmed death ligand 2, CD301b, IFN regulatory factor 4, and moderate upregulation of OX40 ligand. However, thymic stromal lymphopoietin and OX40 ligand were not required for Th2 priming. Thus, our data suggest that appropriate stimuli can induce DC to express the unique signals sufficient to direct CD4(+) T cells to Th2 differentiation.
Subject(s)
Dendritic Cells/immunology , Interleukin-4/biosynthesis , Nippostrongylus/immunology , Th2 Cells/immunology , Animals , Antigens, Ly/biosynthesis , CD11c Antigen/biosynthesis , Cell Differentiation/immunology , Cytokines/genetics , Cytokines/immunology , Green Fluorescent Proteins , Histocompatibility Antigens Class II/biosynthesis , Histocompatibility Antigens Class II/immunology , Interferon Regulatory Factors/biosynthesis , Interleukin-33 , Interleukin-4/immunology , Interleukins/immunology , Larva/immunology , Lectins, C-Type/biosynthesis , Lymph Nodes/immunology , Lymphocyte Activation/immunology , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , OX40 Ligand , Programmed Cell Death 1 Ligand 2 Protein/biosynthesis , Tumor Necrosis Factors/biosynthesis , Tumor Necrosis Factors/immunology , Thymic Stromal LymphopoietinABSTRACT
Hookworms infect more than 700 million people worldwide and cause more morbidity than most other human parasitic infections. Nippostrongylus brasiliensis (the rat hookworm) has been used as an experimental model for human hookworm because of its similar life cycle and ease of maintenance in laboratory rodents. Adult N. brasiliensis, like the human hookworm, lives in the intestine of the host and releases excretory/secretory products (ESP), which represent the major host-parasite interface. We performed a comparative proteomic analysis of infective larval (L3) and adult worm stages of N. brasiliensis to gain insights into the molecular bases of host-parasite relationships and determine whether N. brasiliensis could indeed serve as an appropriate model for studying human hookworm infections. Proteomic data were matched to a transcriptomic database assembled from 245,874,892 Illumina reads from different developmental stages (eggs, L3, L4, and adult) of N. brasiliensis yieldingâ¼18,426 unigenes with 39,063 possible isoform transcripts. From this analysis, 313 proteins were identified from ESPs by LC-MS/MS-52 in the L3 and 261 in the adult worm. Most of the proteins identified in the study were stage-specific (only 13 proteins were shared by both stages); in particular, two families of proteins-astacin metalloproteases and CAP-domain containing SCP/TAPS-were highly represented in both L3 and adult ESP. These protein families are present in most nematode groups, and where studied, appear to play roles in larval migration and evasion of the host's immune response. Phylogenetic analyses of defined protein families and global gene similarity analyses showed that N. brasiliensis has a greater degree of conservation with human hookworm than other model nematodes examined. These findings validate the use of N. brasiliensis as a suitable parasite for the study of human hookworm infections in a tractable animal model.
Subject(s)
Ancylostomatoidea/growth & development , Gastrointestinal Tract/parasitology , Helminth Proteins/metabolism , Life Cycle Stages , Proteome/analysis , Ancylostomatoidea/metabolism , Animals , Base Sequence , Conserved Sequence , Gene Expression Profiling , Gene Expression Regulation, Developmental , Phylogeny , Proteome/metabolism , Proteomics/methods , Rats , Rats, Sprague-Dawley , Sequence Analysis, RNAABSTRACT
A keenly sought therapeutic approach for the treatment of allergic disease is the identification and neutralization of the cytokine that regulates the differentiation of T helper 2 (Th2) cells. Th2 cells are exciting targets for asthma therapies. Recently, the cytokine IL-25 has been shown to enhance Th2-type immune activity and play important roles in mediating allergic inflammatory responses. To investigate this further, we crossed IL-25(-/-) C57BL/6 mice with G4 IL-4 C57BL/6 reporter mice and developed an assay for in vitro and in vivo IL-4-independent Th2-cell differentiation. These assays were used to determine whether IL-25 was critical for the formation of Th2 cells. We found there was no physiological role for IL-25 in either the differentiation of Th2 cells or their development to effector or memory Th2-cell subsets. Importantly, this data challenges the newly found and growing status of the cytokine IL-25 and its proposed role in promoting Th2-cell responses.
Subject(s)
Cell Differentiation , Interleukins/physiology , Th2 Cells/cytology , Animals , Helminthiasis/immunology , Immunologic Memory , Interleukin-4/physiology , Mice , Mice, Inbred C57BLABSTRACT
Exposure to allergens, both man-made and from our environment is increasingly associated with the development of significant human health issues such as allergy and asthma. Allergen induced production of the cytokine interleukin (IL-)4 by Th2 cells is central to the pathogenesis of allergic disease (Gavett et al., 1994). The development of the G4 mouse, that expresses green fluorescent protein (GFP) as a surrogate for IL-4 protein expression has made it possible to directly track the immune cells that produce IL-4. By combining a reliable intradermal immunisation technique with the transgenic G4 mouse we have been able to develop a novel & unique in vivo primary Th2 immune response model (PTh2). When allergens relevant to human disease are evaluated using the PTh2 assay a dose dependent hierarchy of allergenicity is revealed with environmental allergens (cockroach, house dust mite) the most potent and food allergens being the least. In addition, the PTh2 assay is extremely sensitive to the immunoregulatory effects of Mycobacterial extracts and immunosuppressive drugs on primary Th2 cell development. Taken together, this assay provides a standardised method for the identification of the structural and functional properties of proteins relevant to allergenicity, and is a powerful screening tool for novel lead compounds that are effective at inhibiting the primary Th2 response in allergic diseases.