Immune-mediated damage is not essential for the expulsion of Nippostrongylus brasiliensis adult worms from the small intestine of mice

Nippostrongylus brasiliensis adult worms are expelled from the rat small intestine during a primary infection by two steps. First, host immune responses cause damage to the worms, and then a nonspecific inflammatory response initiates expulsion. We have tested the two-step expulsion hypothesis in mice infected with N. brasiliensis. After a primary infection in C57BL/6 mice, adult worms started to lay eggs on day 5 postinfection (p.i.) and were expelled around day 9-10 p.i. According to the rat system, 5 day- and 8-day-old worms were assumed to be 'normal' and 'damaged', respectively. When 5 day- and 8 day-old worms obtained from C57BL/6 mice were transferred surgically into the small intestine of naive C57BL/6 mice, both 5 day- and 8 day-old worms were almost simultaneously expelled by day 6 postworm implantation (p.w.i.). In contrast, when 5 day- and 8 day-old worms of mouse origin were implanted into naive Wistar rats, 8 day-old worms were expelled by day 5 p.w.i., while 5 day-old worms were expelled by day 8 p.w.i. Similar results were obtained when BALB/c mice were used. Therefore, mice can expel N. brasiliensis adult worms as rapidly as rats expel 'damaged' worms, regardless of the status of the worms ('normal' or 'damaged'). Stat6-deficient mice were unable to expel implanted 5 day-old worms up to day 10 p.w.i., suggesting that cellular mechanisms depending on Stat6-signalling system are necessary for the expulsion. When N. brasiliensis adult worms obtained from Stat6-deficient mice 5 and 15 days after a primary infection were implanted into Wistar rats, the former established in the recipient rats for approximately 1 week and were then expelled by day 10 p.w.i., whereas the latter were expelled by day 4 p.w.i. These results suggest that immune-mediated damage of N. brasiliensis adult worms (first step) is not a prerequisite for expulsion from the small intestine of mice, although adult worms are actually damaged by Stat6-independent immune mechanisms.     

Pulmonary inflammation in parasitic infection: immunoglobulins in bronchoalveolar washings of rats infected with Nippostrongylus brasiliensis

Despite marked pulmonary pathology caused by larval stages of many helminth parasites, little is known about the mechanisms of immune and inflammatory responses to parasites in the respiratory tract. Using bronchoalveolar lavage (BAL) we have retrieved soluble proteins and cells from the respiratory tract of rats given a primary or secondary infection with the nematode Nippostrongylus brasiliensis. Total amounts of different immunoglobulin classes and albumin in BAL fluids and serum were quantitated using an ELISA. Analysis of the cellular component showed an increase in alveolar macrophages, neutrophils, eosinophils and lymphocytes on different days post-infection similar to our earlier findings. A time course study revealed that the concentrations of total protein, albumin, IgG, IgA and IgM in BAL fluids of infected animals were increased from days 2 to 32 after a primary infection. The magnitude of this increase was higher following a challenge infection (secondary) with the same parasite. Moreover, there was also a biphasic increase in total protein, IgG and IgA after secondary infections, with peaks on days 2 to 4 and 11 to 21 post-infection. A comparison of immunoglobulin to albumin ratios in serum and BAL fluids showed that the initial peak of proteins in the lavage was a result of serum leakage and the subsequent peak was due to local secretion of immunoglobulins. These results suggest that in addition to marked BAL cellular reactivity, N. brasiliensis infection induces an initial vascular and endothelial permeability in the respiratory tract which is soon repaired but followed by local synthesis and secretion of IgG and IgA in the lower respiratory tract.     

Broncho-alveolar leucocyte responses during primary and secondary Nippostrongylus brasiliensis infection in the rat

Using broncho-alveolar lavage, we have studied the cellular responses in the rat lung following primary and secondary infection with Nippostrongylus brasiliensis. During the primary infection, there was a biphasic increase in total broncho-alveolar leucocytes and in the absolute numbers of macrophages, neutrophils, eosinophils and lymphocytes. The first peak occurred on days 4-6, and the second peak occurred around day 16, after infection. During the secondary infection there was an anamnestic-like response by all cell types. These data suggest that the broncho-alveolar leucocyte responses to infection have an immunological basis and that in addition to the alveolar macrophage, neutrophils, eosinophils and lymphocytes may play a significant role in lung resistance against migrating helminth larvae.     

Chemokine and cytokine expression in murine intestinal epithelium following Nippostrongylus brasiliensis infection

Infection of mice with the nematode parasite Nippostrongylus brasiliensis results in a well characterized intestinal mastocytosis with intraepithelial migration of mucosal mast cells (MMC). The molecules mediating this response are unknown. We examined expression of several putative mast cell chemoattractants in intestinal epithelium following N. brasiliensis infection. Expression of the chemokines monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 alpha (MIP-1alpha), RANTES (regulated on activation normal T-cell expressed and secreted), fractalkine, and thymocyte expressed chemokine (TECK); and the cytokines stem cell factor (SCF) and transforming growth factor beta1 (TGFbeta1), was constitutive and no alteration was detected following infection. MCP-1 expression was also constitutive but at much lower levels and increased expression was detected on days 7 and 14 postinfection. Expression of MCP-1 in whole jejunum was at much higher levels than in epithelium. Constitutive expression of MCP-1, MIP-1alpha and TGFbeta1 was also detected in cultured bone marrow-derived homologues of MMC. In an intestinal epithelial cell line (CMT-93), there was constitutive expression of SCF, TGFalpha1, fractalkine and MCP-1. The results show that, in vivo, epithelium is a potentially important source of mast cell chemoattractants.     

Propulsive activity of the rat small intestine during infection with the nematode Nippostrongylus brasiliensis

Summury Intestinal propulsive motility was measured in rats infected with 4000 Nippostrongylus brasiliensis larvae by following the transit of radioactive chromium (⁵¹Cr) through the gut. On days 6, 8, 10, 12 and 14 post-infection, ⁵¹Cr was injected through an indwelling catheter into the duodenum. The animals were killed 15 min later and the distribution of radioactivity in the small intestine measured. A group of uninfected, catheterized animals served as controls. Intestinal propulsive activity was increased significantly on day 8 post-infection. No significant difference in the overall intestinal transit occurred on days 6,10,12 and 14 post-infection, although it appeared that it may have been decreased in the upper small intestine on day 6. The significance of these results is discussed.     

Studies on chronic versus transient intestinal nematode infections in mice I. A comparison of responses to excretory/secretory (ES) products of Nippostrongylus brasiliensis and Nematospiroides dubius worms

Summary Previous reports have demonstrated that after implantation of intestinal worms or after exposure to infective third stage larvae, the duration of infection with Nematospiroides dubius is markedly prolonged in intact mice relative to infection with Nippostrongylus brasiliensis. The rapid rejection of N. brasiliensis adults appears T-cell dependent in that adults persist for longer periods in hypothymic nude mice than in intact mice. Excretory/secretory (ES) products harvested from N. dubius or N. brasiliensis intestinal worms did not differ obviously in the following characteristics: rate of production and degree of complexity of proteins, in vitro mitogenicity, allergenicity, or in their abilities to induce or elicit delayed type hypersensitivity reactions in naive and infected mice, respectively. Two differences between N. brasiliensis- and N. dubius-infected mice were an IgG₁ hypergammaglobulinaemia and readily detected anti-ES precipitating antibodies in the circulation; both responses were confined to the chronic N. dubius infection. One difference between N. brasiliensis and N. dubius ES products was that the former, but not the latter, induced protection against homologous infection when injected with Freund's complete adjuvant. By contrast, intraperitoneal implantation of either type of adult worm induced protection against homologous infection at least in female Balb/c mice. After intestinal implantation of both N. dubius and N. brasiliensis intestinal worms, the rejection of N. brasiliensis was not influenced by, nor did it alter, persistence of N. dubius adults. In support of conclusions drawn by others, the differences in persistence of infection between these two nematodes probably reflect differences in the ability to resist both specific and nonspecific components of the complex intestinal rejection process. The chronicity of N. dubius infection and nonpersistence of N. brasiliensis     

Toxocara canis larval excretory/secretory proteins impair eosinophil-dependent resistance of mice to Nippostrongylus brasiliensis

Survival of parasitic helminths within a host requires immune evasion and excretory/secretory (ES) proteins may contribute to this process. Eosinophils are important effector cells in immunity of mice to the nematode Nippostrongylus brasiliensis and eosinophilic interleukin-5 transgenic (IL-5 Tg) mice are highly resistant to the earliest stages of primary infections. In contrast, Toxocara canis is largely resistant to eosinophils, with viable larvae encysted in tissues often surrounded by these and other leucocytes. The aim of this study was to investigate whether T. canis ES (TES) proteins inhibit eosinophil-dependent resistance to N. brasiliensis . Mouse serum pre-treated with TES had reduced capacity to mediate the adherence of leucocytes to N. brasiliensis infective-stage larvae (L3) and this correlated with reduced complement C3 deposition on the parasite. TES did not inhibit eosinophil survival or eotaxin-dependent eosinophil migration in vitro . Cellular inflammation and eosinophil degranulation in the skin in response to injection of L3 was also not impaired by TES. However, when TES was included with L3 in an inoculum given to IL-5 Tg mice, a greatly increased number of parasites migrated to the lung. This suggests that the early eosinophil-dependent resistance in these mice was suppressed, by mechanisms yet to be determined.     

Involvement of complement and fibronectin in eosinophil-mediated damage to Nippostrongylus brasiliensis larvae

By using IL-5 transgenic mice, it has been shown that eosinophils might play a key role in elimination of larval stages of nematode infections. The present study was carried out to clarify molecular mechanisms involved in the eosinophil-mediated killing of Nippostrongylus brasiliensis larvae. The larvicidal activity was observed in the presence of normal serum in vitro. Electron microscopic observations revealed firm attachment of eosinophils to the cuticular surface of larvae, which was damaged by electron-dense materials released from eosinophils. The larvicidal activity was abrogated by heat- or zymosan-treatment of the serum, whereas depletion of IgG or IgM from the serum did not interfere with eosinophil adhesion and killing. Moreover, pretreatment of eosinophils with monoclonal antibodies against CD11b or VLA-4 inhibited the eosinophil-mediated killing of larvae. Immunofluorescent staining demonstrated the deposition of C3c and plasma fibronectin on the cuticle of the larvae. These results indicate that interactions between CD11b and VLA-4 and their respective counter-ligands deposited on the cuticle are essential in eosinophil-mediated adhesion and damage to larvae of N. brasiliensis.     

Differentially enhanced cytokine gene expression in CD4+ and CD8+ T cells in mesenteric lymph nodes in rats infected with Nippostrongylus brasiliensis

Autoinfective strongyloidiasis is potentially fatal, yet the majority of infected individuals harbour asymptomatic and chronic infections. The role of humoral responses in modulating autoinfection was assessed by examining antibody isotype responses to filariform larval antigens amongst chronically infected ex-Far East Prisoners of War (exFEPOWs) with longstanding (> 30 years) infection. Serum immunoglobulin (Ig)G1, IgG4, IgE and IgA responses to whole Strongyloides stercoralis L3 extracts and their constituent antigenic components were characterized by ELISA and quantitative immunoblotting. Comparison of two groups of S. stercoralis infected exFEPOWs with and without detectable larvae in stool demonstrated novel trends. Significantly enhanced recognition of six immunodominant antigenic components by IgA was associated with undetectable larval output, as was enhanced IgE recognition of several components. Additionally, IgE and IgG4 exhibited parallel antigen recognition patterns. These findings are consistent with roles for IgA in modulating larval output, for IgE in regulating autoinfection, and for IgG4 in blocking IgE-mediated responses in human strongyloidiasis.     

Type 2-biased expression of cytokine genes in lung granulomatous lesions induced by Nippostrongylus brasiliensis infection

Infections with helminthic parasites occasionally induce pulmonary diseases with possible involvement of immunological mechanisms. In rats infected with the nematode Nippostrongylus brasiliensis, pulmonary granulomatous lesions develop and persist after the larvae have migrated through the lungs. To determine the pathogenesis of this lesion, we examined cytokine gene expression in the lungs using RT-PCR and in situ hybridization. Two weeks after infection, when fully developed lesions appeared, levels of IL-3 and of type2 cytokines IL-4, IL-5, IL-6 and IL-13 gene expression were markedly enhanced in whole lung homogenates. Those of IL-2 and IFN-gamma were also slightly increased 2 weeks postinfection. IL-12 mRNA level did not change after 2 weeks but was slightly increased after 4 weeks. Levels of IL-10 and proinflammatory cytokine TNF gene expression did not show significant changes, although a slight increase was observed in IL-1beta message after 2 weeks. In situ hybridization studies showed that lung granulomatous lesions were composed mainly of lymphoid cells expressing IL-3, IL-4 and IL-13 mRNA, but not IFN-gamma mRNA. IL-5 mRNA-expressing cells were fewer in number than these cells. RMCP II immunohistochemistry revealed that mast cells increased in number in the lung granulomas. From these results, it was concluded that the nematode infection-associated lung granuloma was a type 2 lesion.     

Different susceptibility to the IL-3 induced-protective effects between Strongyloides ratti and Nippostrongylus brasiliensis in C57BL/6 mice

Repetitive administration of recombinant IL-3 induced protection against Strongyloides ratti but not against Nippostrongylus brasiliensis in C57BL/6 mice. Numbers of S. ratti were negligible from day 4 to day 6 post-infection in mice injected with IL-3, whereas N. brasiliensis burdens were almost equal from day 4 to day 6 between mice injected with IL-3 or with medium. Mice treated with IL-3 and then concurrently infected with S. ratti and N. brasiliensis were protected from intestinal S. ratti but not from N. brasiliensis. The numbers of intestinal mucosal mast cells were increased by the repetitive IL-3 treatment on one day after the final injection and was augmented by subsequent infection with both nematodes.     

The detection and measurement of coproantibodies to Nippostrongylus brasiliensis in rats following a primary infection

Summary Investigations were initiated to study the possible detection and measurement of coproantibodies in animals infected with a gastrointestinal nematode parasite. Faecal extracts, extracts of small intestinal mucosa and sera of rats infected with intestinal nematode Nippostrongylus brasiliensis were examined for total IgA, IgM and IgG levels and haemagglutinating and precipitating antibodies specific to parasite antigens over a 30-day-period following infection. It was found that in both faecal and mucosal extracts immunoglobulin concentrations increased after a primary infection. In faecal extracts there was a seven-fold increase of IgA, a three to six-fold increase of IgG and about a fifty-fold increase of IgM. Haemagglutinins in faecal extracts detected by adult worm excretory-secretory (ES) products and adult worm and infective larvae somatic extracts were observed from 3 days after infection (DAI). Haemagglutinins detected by ES products reached their highest titres on 11–12 DAI while those reacting with adult worm somatic extracts showed the highest level between IS and 19 DAI. A similar pattern of response was found in the antibody levels of the intestinal mucosa. Haemagglutinins detected in faeces during the first 12 DAI reacted with the same antigens as antibodies present in the sera at that time but coproantibodies from 18, 24 and 30 DAI were different from those circulating in sera at that stage of the infection. The results suggest that measurement of coproantibody levels may provide a convenient and useful index of local immune responses to gastrointestinal helminths.     

Kinetics of expulsion of the nematode, Nippostrongylus brasiliensis, in mast-cell deficient W/Wv mice

Mucosal mast-cell hyperplasia is frequently observed in intestinal nematode infections and it has been suggested that mast-cell responses to parasite antigens are involved in worm expulsion (self cure). To evaluate the importance of this mechanism, the course of infection and expulsion of Nippostrongylus brasiliensis was compared in mast-cell deficient W/WV and normal (+/+) mice. Initial infectivity rates were similar, but the subsequent kinetics of expulsion of adult worms differed principally in that the onset of expulsion in mast-cell deficient mice appeared to occur 24-36 h later than that in normal mice. Expulsion was complete by the 14th day post infection in both W/WV and normal mice. Worm fertility (as estimated by faecal egg output) also differed in W/WV and normal mice, with maximal egg output in W/WV mice occurring 24 h later than that in normal mice. Although a few mast cells were present in the intestinal mucosa and tongue of W/WV mice, their numbers did not change during the course of infection with N. brasiliensis. In contrast, worm expulsion in normal mice was associated with a moderate increase in numbers of intestinal mast cells, commencing at the onset of expulsion and peaking several days after expulsion was completed.     

Free radical generation during primary infections with Nippostrongylus brasiliensis

The production of free radicals during infection of the rat with Nippostrongylus brasiliensis was investigated. Lipid peroxidation, which is the best documented effect of free radicals, was monitored in the small intestines of infected rats by measurement of malonyldialdehyde and was found to be increased at the time of worm rejection. The capacity of peritoneal leucocytes to produce free radicals, as measured by chemiluminescence, was monitored in rats infected with different doses of N. brasiliensis. Rejection of N. brasiliensis from rats infected with 6000 third-stage larvae (L3) began 2 days earlier than in rats infected with only 600 L3. Maximal free radical generation also occurred 2 days earlier and was quantitatively greater in rats infected with 6000 L3. Free radical generation by leucocytes in response to live adult N. brasiliensis was enhanced by plasma from infected rats indicating the existence of a plasma-borne factor responsible for the initiation of free radical generation in response to N. brasiliensis.     

Pulmonary immune responses to Nippostrongylus brasiliensis: isotype-specific antibodies in bronchoalveolar lavage fluids of rats

Larvae of Nippostrongylus brasiliensis have an obligatory migratory phase through the lungs of rats during their development. Since earlier studies have shown that this migration is associated with accumulation of Fc receptor bearing effector cells in the bronchoalveolar spaces, wc have analysed antibody reactivity in bronchoalveolar lavage fluids (BALF) during development of immune responses against N. brasiliensis. The development of parasite specific antibodies in bronchoalveolar spaces was similar to that in the serum, but was of a lower titre. A secondary infection resulted in an anamnestic response. Iso type analysis showed that IgG, IgA and IgM antibodies were present in BALF and they recognized several proteins of the parasite ranging from 16-290 kDa. Immunoblot analysis on two-dimensional electrophoretic separated parasitic proteins identified stage specific differences in the BALF antibody responses. IgG was the predominant class of antibody in BALF and when compared with serum, IgM antibody responses were weak. Thus, infection with N. brasiliensis resulted in the appearance of site-, stage- and isotype-specific antibody responses in the lungs of rats.     

The heterolgous protection of rats against a challenge with Fasciola hepatica by prior infection with the nematode Nippostrongylus brasiliensis

Previous infection of rats with Nippostrongylus brasiliensis was shown to result in protection against an oral challenge with Fusciola hepatica metacercariae but not against an intraperitoneal challenge with newly excysted juvenile (NEJ) flukes. The timing of the challenge was important and a double infection with the nematode gave more consistent results than a single. Resistance appeared to be associated with a prior induction of intestinal eosinophilia. Sera from these resistant rats, however, failed to induce eosinophil adherence to NEJ flukes in vitro.     

巴西日圆线虫虫体蛋白体外诱导THP-1来源巨噬细胞极化的研究

目的　分析巴西日圆线虫虫体蛋白体外诱导人单核细胞白血病THP?1细胞来源巨噬细胞的极化方向,探索机体对巴西日圆线虫感染的免疫应答机制。方法　建立并维持巴西日圆线虫培养的实验室循环,无菌条件下收集L3和L5虫体,分别制备虫体蛋白;建立THP?1细胞体外培养体系,经500 ng/mL佛波酯（PMA）刺激培养后呈贴壁状态的M0型细胞分别采用500 ng/mL脂多糖（LPS）+ 100 ng/mL γ?干扰素（IFN?γ）、白细胞介素4（IL?4）+ IL?13（浓度均为100 ng/mL）、L3及L5虫体蛋白（浓度均为5 mg/mL）进行刺激,同时设不加任何刺激的阴性对照组。通过显微镜镜检观察刺激后细胞形态、实时荧光定量PCR（qPCR）检测M1/M2型巨噬细胞特异性基因mRNA表达水平、流式细胞术检测巨噬细胞表面标志物及酶联免疫吸附试验（ELISA）检测M1/M2型巨噬细胞分泌的细胞因子含量,观察巴西日圆线虫虫体蛋白体外诱导THP?1来源巨噬细胞的极化方向。结果　THP?1细胞经PMA刺激培养呈贴壁的M0型细胞后,经LPS + IFN?γ刺激培养后呈特征性M1型极化;经IL?4 + IL?13刺激培养后呈特征性M2型极化;经L3和L5蛋白刺激培养后均呈特征性M2型极化。阴性对照组、LPS + IFN?γ刺激组、IL?4 + IL?13刺激组、L3蛋白刺激组、L5蛋白刺激组间M1型巨噬细胞比例差异有统计学意义（[χ2] = 3 721.00,P < 0.001）,其中LPS + IFN?γ刺激组M1型巨噬细胞比例最高;各组间M2型巨噬细胞比例差异有统计学意义（[χ2] = 105.43,P < 0.001）。各组间C?C基序趋化因子配体2（CCL2）、肿瘤坏死因子α（TNF?α）、IL?12b、过氧化物酶体增殖物激活受体γ（PPARγ）、IL?10、甘露糖受体C型1（Mrc1）基因mRNA表达水平差异均有统计学意义（F = 191.95、129.95、82.89、11.30、9.51、12.35,P均< 0.001）,各组间CD86、CD206阳性率差异均有统计学意义（[χ2] = 24 004.33、832.50,P均< 0.001）。LPS + IFN?γ刺激组IL?1β、TNF?α表达水平均显著高于IL?4 + IL?13刺激组、L3蛋白刺激组及L5蛋白刺激组（P均< 0.001）;IL?4 + IL?13刺激组、L3蛋白刺激组和L5蛋白刺激组TGF?β1、IL?10表达水平均显著高于阴性对照组和LPS + IFN?γ刺激组（P均< 0.05）。结论　巴西日圆线虫L3和L5虫体蛋白体外均可诱导THP?1来源巨噬细胞向M2型极化。     

Reduced amount of intestinal mucus by treatment with anti-CD4 antibody interferes with the spontaneous cure of Nippostrongylus brasiliensis-infection in mice

Mechanism of spontaneous cure was studied in mice infected with mouse-nonadaptive Nippostrongylus brasiliensis. Adult BALB/c mice were cured spontaneously of infection with this strain of N. brasiliensis by Day 7 post-infection. Expulsion of intestinal worms was delayed dose-dependently by a treatment with anti-CD4 antibody. However, the treatment had no significant effect on larval recovery from the lungs. Treatment of mice with anti-IL-5 antibody suppressed intestinal tissue eosinophilia induced by the infection, but did not affect intestinal worm recovery. Antigen specific IgE antibody was not detected in the sera obtained from Days 5 to 15. Therefore, IL-5 and specific IgE antibody are probably not important in the spontaneous cure. Treatment of mice with anti-CD4 antibody had no significant effect on number of intestinal goblet cells or on expression of terminal sugars of goblet cell mucins. However, histological and quantitative analyses revealed that significantly less intestinal mucus was released in anti-CD4 antibody treated mice than in control mice. These results suggest that CD4+ lymphocytes control the amount of intestinal mucus and consequently the reduced mucus interferes with the spontaneous cure. Quantity of mucus released in the intestinal lumen may have an essential role in the spontaneous cure ofN. brasiliensis-in/fcfio/i of mice.