RESUMO
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.
Assuntos
Interleucina-9 , Nippostrongylus , Camundongos , Animais , Nippostrongylus/fisiologia , Interleucina-9/metabolismo , Imunidade Inata , Citocinas/metabolismo , Linfócitos T CD4-PositivosRESUMO
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.
Assuntos
Infecções por Nematoides , Doenças dos Roedores , Animais , Mastócitos , Molibdênio , Infecções por Nematoides/veterinária , Nippostrongylus/fisiologia , Peptídeo Hidrolases , RatosRESUMO
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.
Assuntos
Memória , Células Mieloides/citologia , Nippostrongylus/fisiologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/fisiopatologia , Animais , Cognição , Macrófagos/citologia , Aprendizagem em Labirinto , Camundongos , Microglia/patologia , Infecções por Strongylida/patologiaRESUMO
Hookworm infections (Necator americanus or Ancylostoma duodenale) represent a major neglected tropical disease, affecting approximately 700 million people worldwide, and can cause severe morbidity due to the need for these worms to feed on host blood. N. brasiliensis and H. polygrus, both rodent parasites, are the two most commonly employed laboratory models of experimental hookworm infection. Both parasites evoke type 2 immune responses, and their use has been instrumental in generating fundamental insight into the molecular mechanisms of type-2 immunity and for understanding how the immune response can control parasite numbers. Here we provide a complete set of methods by which to investigate the natural progression of infection and the host immunological responses in the lung and intestine of H. polygyrus- and N. brasiliensis-infected mice. Detailed information is included about the most important parasitological and immunological measurements to perform at each time point. © 2017 by John Wiley & Sons, Inc.
Assuntos
Modelos Animais de Doenças , Imunidade Humoral , Camundongos , Nematospiroides dubius/fisiologia , Nippostrongylus/fisiologia , Infecções por Strongylida/imunologia , Animais , Progressão da DoençaRESUMO
Mucin is a major component of mucus in gastrointestinal mucosa. Increase of specific sialomucins having Sda blood group antigen, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß-, is considered to be associated with expulsion of the parasitic intestinal nematode Nippostrongylus brasiliensis. In this study, we examined the relationship between interleukin (IL)-13 pathway and expression of Sda-sialomucins in small intestinal mucosa with N. brasiliensis infection. Nematode infection induced marked increases in small intestinal mucins that reacted with anti-Sda antibody in wild type (wt) mice. However, this increase due to infection was supressed in IL-4 receptor α deficient (IL-4Rα-/-) mice, which lack both IL-4 and IL-13 signaling via IL-4R, and severe combined immunodeficient (SCID) mice, which have defects in B- and T-lymphocytes. Analysis using tandem mass spectroscopy showed that Sda-glycans were not expressed in small intestinal mucins in IL-4Rα-/- and SCID mice after infection despite the appearance of Sda-glycans in the infected wt mice. Inoculation of recombinant IL-13 into the infected SCID mice restored expression of Sda-glycan. Our results suggest that the IL-13/IL-4R axis is important for the production of Sda-sialomucins in the host intestinal mucosa with parasitic nematode infection.
Assuntos
Imunidade Adaptativa , Enteropatias Parasitárias/imunologia , Mucosa Intestinal/imunologia , Receptores Tipo II de Interleucina-4/genética , Sialomucinas/metabolismo , Infecções por Strongylida/imunologia , Animais , Enteropatias Parasitárias/parasitologia , Intestino Delgado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Nippostrongylus/fisiologia , Receptores Tipo II de Interleucina-4/metabolismo , Transdução de Sinais , Infecções por Strongylida/parasitologiaRESUMO
The present study investigates the in vitro efficacy of derivatives of the cyclooctadepsipeptides and the aminophenylamidines, which are promising candidates for the evaluation of the treatment of human soil-transmitted helminthiases. The effects of emodepside and PF1022A as well as of amidantel, deacylated amidantel and tribendimidine were evaluated in a concentration range between 0.01 and 100 µg/ml against third-stage larvae (L3) and adult worms of Nippostrongylus brasiliensis and first-stage larvae (L1) of Trichinella spiralis. Furthermore, drug combinations of PF1022A plus deacylated amidantel or tribendimidine and of tribendimidine plus levamisole were tested for any potential additive or even synergistic interactions. Emodepside had a significantly lower EC(50) value than PF1022A in the T. spiralis (0.02788 vs. 0.05862 µg/ml) and the N. brasiliensis (0.06188 vs. 0.1485 µg/ml) motility assays but not in the acetylcholine esterase secretion assay with adult N. brasiliensis (0.05650 vs. 0.06886 µg/ml). While amidantel showed only minimal or at best partial inhibition of nematode motility and acetylcholine esterase secretion, tribendimidine was nearly as potent as deacylated amidantel. Whereas deacylated amidantel had a significantly lower EC(50) than tribendimidine in the N. brasiliensis L3 motility assay (0.05492 vs. 0.2080 µg/ml), differences were not significant in the T. spiralis L1 motility assay (0.7766 vs. 1.145 µg/ml). Surprisingly, none of the combinations showed improved efficacy when compared to the individual drugs including levamisole/tribendimidine, which have previously been reported to act synergistically against Ancylostoma ceylanicum.
Assuntos
Anti-Helmínticos/farmacologia , Depsipeptídeos/farmacologia , Nippostrongylus/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Trichinella spiralis/efeitos dos fármacos , Animais , Sinergismo Farmacológico , Larva/efeitos dos fármacos , Larva/fisiologia , Locomoção/efeitos dos fármacos , Nippostrongylus/fisiologia , Trichinella spiralis/fisiologiaRESUMO
Infections with the parasitic helminth, Nippostrongylus brasiliensis, cause changes in rat small intestinal goblet cell mucin, particularly in the peripheral sugar residues of oligosaccharide. These changes may correlate with expulsion. In this study, we examined changes in mucin oligosaccharides caused by primary infection and reinfection with N. brasiliensis, using two monoclonal antibodies, HCM31 and PGM34, that react with sialomucin and sulfomucin, respectively. Enzyme-linked immunosorbent assay of jejunal mucins showed that the relative reactivity of mucins with HCM31, but not PGM34, increased up to 16 days after primary infection and 6 days after reinfection, the times when the worms were expelled from the rats. Immunohistochemical studies confirmed that goblet cells stained with HCM31 greatly increased at the time of worm expulsion. These results indicate that the marked increase observed in HCM31-reactive sialomucins may be related to expulsion of the worms.
Assuntos
Enteropatias Parasitárias/metabolismo , Jejuno/metabolismo , Nippostrongylus/fisiologia , Sialomucinas/metabolismo , Infecções por Strongylida/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Células Caliciformes/metabolismo , Imunidade nas Mucosas , Imuno-Histoquímica , Enteropatias Parasitárias/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Jejuno/parasitologia , Cinética , Lectinas , Masculino , Nippostrongylus/imunologia , Contagem de Ovos de Parasitas , Ratos , Ratos Wistar , Sialomucinas/imunologia , Infecções por Strongylida/imunologiaRESUMO
Expulsion of adult Nippostrongylus brasiliensis worms from the small intestine is profoundly impaired in signal transducer and activator of transcription (STAT)6-deficient mice. IL-5 transgenic (Tg) mice with constitutive eosinophilia show profound early resistance in the skin and/or later pre-lung phase of primary infections with N. brasiliensis. This study was designed to assess the importance of the eosinophil chemokine eotaxin and the STAT6/interleukin (IL)-4/IL-13 signalling pathway in early resistance to N. brasiliensis. Eosinophil recruitment into the skin following injection of N. brasiliensis larvae was reduced in STAT6- or eotaxin-deficient/IL-5 Tg double mutant mice. While ablation of eotaxin did not impair resistance in the pre-lung phase of N. brasiliensis infections in IL-5 Tg mice, elimination of STAT6 caused a modest reduction in resistance in both primary and secondary infections on this genetic background. STAT6(-/-)-, IL-13(-/-)- and IL-4Ralpha(-/-)-deficient single mutant and IL-13(-/-)/IL-4Ralpha(-/-) double mutant mice were more susceptible than WT mice during the pre-lung phase of secondary N. brasiliensis infections. In contrast, primary or secondary resistance were unaffected at either the pre-lung or gut stages of infection in eotaxin(-/-) single mutant mice. STAT6(-/-) and eotaxin(-/-) mice with or without the IL-5 transgene, were no more susceptible than WT or IL-5 Tg mice to protracted primary infections with Heligmosomoides bakeri, a parasitic nematode that is restricted to the gut. Our data suggest that parasitic nematodes that transit through the skin and lungs en route to the gut may be susceptible to early (pre-lung) innate and adaptive immune mechanisms that are dependent on the STAT6/IL-4/IL-13 signalling pathway, and this may be important for the development of effective therapies and vaccines.
Assuntos
Quimiocina CCL11/fisiologia , Eosinófilos/metabolismo , Heligmosomatoidea/fisiologia , Nippostrongylus/fisiologia , Fator de Transcrição STAT6/fisiologia , Transdução de Sinais/fisiologia , Animais , Quimiocina CCL11/deficiência , Quimiocina CCL11/genética , Eosinófilos/citologia , Eosinófilos/parasitologia , Fezes/parasitologia , Feminino , Interações Hospedeiro-Parasita , Imunidade Inata , Interleucina-5/genética , Interleucina-5/metabolismo , Interleucina-5/fisiologia , Mucosa Intestinal/metabolismo , Intestinos/parasitologia , Larva/fisiologia , Pulmão/metabolismo , Pulmão/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Contagem de Ovos de Parasitas , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Fator de Transcrição STAT6/deficiência , Fator de Transcrição STAT6/genética , Pele/metabolismo , Pele/parasitologiaRESUMO
Plasma butyrylcholinesterase (BChE) hydrolyzes ester-containing compounds such as succinylcholine, as well as acting as a scavenger against neurotoxic organophosphates (OPs). We previously found that Nippostrongylus brasiliensis infection makes rats more susceptible to OP toxicity by decreasing serum paraoxonase-1 (PON1) activity. In the present study, we examined the effects of N.brasiliensis infection on acetylcholinesterase (AChE) activity in plasma, red blood cells (RBCs), brain and diaphragm, as well as serum PON1 activity, in rats at day 7 after infection. N.brasiliensis infection significantly decreased plasma BChE and PON1 activities without significantly altering AChE activity in RBCs, brain and diaphragm. These results provide further insight into the unusual deleterious effects of intestinal nematode infections on body homeostasis.
Assuntos
Butirilcolinesterase/sangue , Nippostrongylus/fisiologia , Infecções por Strongylida/enzimologia , Acetilcolinesterase/análise , Acetilcolinesterase/sangue , Animais , Arildialquilfosfatase/sangue , Encéfalo/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Diafragma/enzimologia , Eritrócitos/enzimologia , Fezes/parasitologia , Masculino , Contagem de Ovos de Parasitas , Distribuição Aleatória , Ratos , Ratos Wistar , Infecções por Strongylida/sangueRESUMO
Nippostrongylus brasiliensis larvae are particularly susceptible to immunological attack during the pre-lung stage of primary and secondary infections in mice. Whilst most of the common laboratory strains of mice are permissive hosts for the parasite, in this study we report for the first time, the strong resistance of naive FVB/N mice to N. brasiliensis. Damage to larvae is evident within the first 24 h of infection and this may be critical to later larval development and reproductive success. Inflammatory responses in the skin, and larval escape from this tissue were comparable in susceptible CBA/Ca and resistant FVB/N mice, with most larvae exiting within 4 h of a primary infection. Lung larval burdens were also similar between strains, but larvae recovered from FVB/N mice were smaller and less motile. In FVB/N mice, larval colonization of the gut was impaired and worms produced very few eggs. However FVB/N mice did not show enhanced resistance to Heligmosomoides bakeri (also known as Heligmosomoides polygyrus), a nematode largely restricted to the gut. Damage done in the pre-lung or lung stages of infection with N. brasiliensis is likely to contribute to ongoing developmental and functional abnormalities, which are profoundly evident in the gut phase of infection.