NK1.1+ cell depletion in vivo fails to prevent protection against infection with the murine nematode parasite Trichuris muris

Protection against the murine nematode parasite Trichuris muris has been shown to involve interleukin 4 (IL-4). NK1.1+ T cell receptor alphabeta+ cells, designated Natural Killer T (NKT) cells, produce a large amount of IL-4 in response to anti-CD3 stimulation and numerous pieces of evidence suggest that NKT cells provide the initial source of IL-4 for T helper 2 (Th2) priming. These observations allow the hypothesis that NKT cells produce a large amount of IL-4 in response to T. muris infection and augment Th2 responses and IL-4 production, thus achieving protection against T. muris. To investigate the involvement of NKT cells in protection against T. muris infection, NK1.1+ cell-depleted B10.BR mice were prepared by anti-NK1.1 monoclonal antibody injection. Efficient expulsion of T. muris worms occurred in NK1.1+ cell-depleted infected mice, and the expulsion kinetics of T. muris worms, the levels of IL-4 production by mesenteric lymph node cells, and the kinetics of the specific IgG1 and IgG2a responses to T. muris were similar to those in mouse IgG-treated or non-treated control B10.BR mice. These observations suggest that NK1.1+ cells and NKT cells are not involved in the induction of Th2 responses and protective immunity to T. muris infection.     

Modulation of cytokine production and response phenotypes in murine trichuriasis

BALB/K mice are usually resistant to infection with the intestinal nematode parasite Trichuris muris and exhibit a Th2 dominated (IL-5, IL-9) response. Conversely in B10.BR mice, which are unable to expel T. muris, Th1 type (IFN-gamma producing) cells predominate. We have manipulated the course of infection in these two strains of mice such that the period of host-parasite contact is extended in the former and curtailed in the latter. Extension of host-parasite contact in BALB/K mice beyond normal (day 21) resulted in the modulation of cytokines produced by in vitro concanavalin A (Con-A) stimulated MLNC away from IL-5 and IL-9 (Th2-type cytokines) in favour of the Th1-type cytokine IFN-gamma. Curtailment of host parasite contact in B10.BR mice to less than 21 days resulted in elevated production of IL-5 and IL-9 by MLNC in the absence of elevated IFN-gamma levels. Thus modulation of expulsion phenotype also modulates cytokine production by T-cells in the MLN draining the site of infection, with a Th2 response being associated with resistance and a Th1 type response with the inability to expel the parasite. Mechanisms by which the modulated cytokine profiles arise are discussed.     

Immunological relationships during primary infection with Heligmosomoides polygyrus (Nematospiroides dubius): downregulation of specific cytokine secretion (IL-9 and IL-10) correlates with poor mastocytosis and chronic survival of adult worms

Mice were infected either with Trichinella spiralis (day 0). Heligmosomoides polygyrus (day – 14) or concurrently with both species and were killed in groups, together with naïve control mice, on 2 occasions (day 8 and 15 post infection with T. spiralis, corresponding to days 22 and 29 p.i. with H. polygyrus). The expulsion of T. spiralis was slowed significantly in concurrently infected mice and this was associated with a reduced mastocytosis and lower serum mucosal mast cell protease levels. Mesenteric lymph node (MLN) lymphocytes from all three experimental groups secreted IL-3 and IL-4 in copious amounts when stimulated in vitro by Concanavalin A (Con-A), but the secretion of high levels of IL-9 and IL-10 was essentially confined to mice infected with T. spiralis alone. It is suggested that adult H. polygyrus selectively modulate cytokine secretion by Th2 cells within the MLN during infection and that this is brought about as a direct consequence of the mechanism employed by H. polygyrus to depress mucosal inflammatory responses in order to facilitate its own survival.     

Comparative studies on immune responses to infection in susceptible B10.BR mice infected with different strains of the murine nematode parasite Trichuris muris

A comparison of immune responses to infection between groups of B10.BR mice infected with different strains of T. muris , S strain (isolated in Sobreda, Portugal), E strain (isolated in Edinburgh), and E-J strain (originally E strain, which has been maintained in our laboratory, Japan), was performed. In mice infected with E and E-J strains, most of the worms were expelled by day 32 after infection, though the expulsion was faster in E-J strain-infected mice. In contrast, no expulsion was observed in S strain-infected mice by day 32 and egg production occurred on day 32. IL-4 production occurred in concanavalin A (Con-A)-stimulated mesenteric lymph node cells (MLNC) from B10.BR mice infected with E and E-J strains, whereas no IL-4 production was observed in S strain-infected mice. IL-4 production did not occur in normal mice. In comparison with normal mice, high levels of IFN-γ production by Con A-stimulated MLNC were detected in mice infected with every strain of  T. muris . IFN-γ production in S strain-infected mice was greater, occurred earlier and was more persistent than in mice infected with E and E-J strains. IgG1 and IgG2a antibodies to T. muris excretory/secretory antigens were observed in B10.BR mice infected with every strain of T. muris . Antibody production showed similar kinetics. These differences in the expulsion kinetics and IL-4 production in B10.BR mice infected with S, E, and E-J strains suggest the involvement of IL-4 in protection against T. muris infection, and confirm the previous conclusion by Else et al . (1994).     

旋毛虫感染小鼠IgG、IL-2和T淋巴细胞亚群动态变化的观察

目的　了解小鼠感染旋毛虫后 Ig G抗体水平、IL - 2分泌量和 T淋巴细胞亚群的动态变化。方法　分别于旋毛虫感染后第 7、14、2 1、2 8和 35天 ,采用 EL ISA方法检测血清特异性 Ig G抗体水平和 IL- 2含量 ,采用流式细胞仪检测 CD4+、CD8+T细胞百分率。结果　 Ig G抗体水平在感染后逐渐上升 ,感杂后 35天达最高值 ;T淋巴细胞的变化表现为 CD4+T细胞减少、CD8+T细胞增多 ,CD4+/ CD8+细胞比值下降 ,以感染后第 14天最为显著 ,直到感染后第 35天仍未见恢复。IL - 2分泌量以感染后第 7天达高峰 ,然后迅速下降 ,到感染后第 35天低于正常组。讨论　旋毛虫感染的急性期 ,小鼠出现免疫抑制现象。抗旋毛虫感染的保护性免疫是由细胞免疫与体液免疫协同完成的。     

Protection in lambs vaccinated with Haemonchus contortus antigens is age related, and correlates with IgE rather than IgG1 antibody

The involvement of mucosal mast cells (MMC) in protection against infection with the murine nematode parasite Trichuris muris was studied in genetically mast cell-deficient WBB6F1-W/Wv mice and their normal littermates WBB6F1-+/+ mice. Expulsion of T. muris worms occurred in infected +/+ mice, whereas no worm expulsion was observed in infected W/Wv mice where the infection persisted until at least day 46 postinfection. No MMC responses were induced in either infected W/Wv or +/+ mice. Specific IgG1and IgG2a antibodies to T. muris excretory/secretory antigens were observed in infected W/Wv and +/+ mice, and antibody production showed similar kinetics. Interleukin 4 production by concanavalin A (Con A)-stimulated mesenteric lymph node cells (MLNC) was induced preferentially in infected +/+ mice. T. muris infection increased the levels of IFN-gamma produced by Con A-stimulated MLNC of infected W/Wv and +/+ mice, with the levels of IFN-gamma in infected W/Wv mice being higher than those in infected +/+ mice. Taken together, these results indicate that W/Wv and +/+ mice are susceptible and resistant to T. muris infection, respectively, and that MMC responses are not required for protective immunity.     

Protective CD4+ T-cell lines raised against Plasmodium chabaudi show characteristics of either Th1 or Th2 cells

Splenic T-lymphocyte lines were established in vitro from Plasmodium chabaudi-infected NIH mice on days 16 and 20 of a primary infection, and from mice after two or three infections. Each line responded specifically to stimulation with a lysed soluble extract of P. chabaudi-infected erythrocytes (pRBC), and displayed a CD4⁺ (L3T4⁺) surface phenotype. Both the day 16 and 20 cell lines, when stimulated in vitro, secreted interleukin-2 (IL-2) and γ-interferon (IFN-γ), indicative of their belonging to the T helper 1 (Th1) CD4⁺ subset. In contrast, both lines derived from reinfected mice secreted interleukin-4 (IL-4) and provided helper activity in antibody production to P. chabaudi in vitro, and thereby had the characteristics of Th2 cells. All four T-cell lines provided significant protection to naïve mice infected with P. chabaudi. In immuno-compromised mice, the day 16 T-cell line was as protective as in naïve mice whereas the cell line from mice infected twice required the additional transfer of mature naïve splenic B cells to provide protection comparable to that seen in the immunocompetent naïve recipients. The results establish that protective immunity to P. chabaudi may be associated with the induction of CD4⁺ Tcells of either the Th1 or Th2 subset which confer protection against this malaria parasite by mechanisms independent of, and dependent upon, B-cell involvement, respectively.     

Contrasting effects of Trichinella spiralis and Trichuris muris antigens on the infection by Leishmania infantum in BALB/c mice

The interaction of Trichinella spiralis and Trichuris muris derived antigens with the infection by Leishmania infantum was investigated in BALB/c mice. Infection with 10(6) promastigotes of L. infantum did not induce relevant serum antibody (IgG subclasses), nor cytokine (IFN-gamma, IL-4) responses despite that mice could partially control the infection. Immunization with T. spiralis activated a moderate IgG1 and secondarily an IgG2a anti-leishmanial response whereas immunization with T. muris elicited only a weak and late activation of IgG1 anti-leishmanial response. Immunization with T. muris caused an elevation of serum IFN-gamma levels which was drastically reinforced by the L. infantum infection, and that was accompanied by almost complete parasitological cure of infected mice. Immunization with T. spiralis induced an elevation of serum IL-4 levels but this response was greatly (about 60%) neutralized by the infection with L. infantum, and this was associated to exacerbation of the infection.     

Mouse mast cell protease-1 is required for the enteropathy induced by gastrointestinal helminth infection in the mouse

BACKGROUND & AIMS: The relationship between intestinal pathology and immune expulsion of gastrointestinal nematodes remains controversial. Immune expulsion of gastrointestinal helminth parasites is usually associated with Th2 responses, but the effector mechanisms directly responsible for parasite loss have not been elucidated. Mast cell hyperplasia is a hallmark of infection with gastrointestinal nematodes, in particular Trichinella spiralis. Although the precise mechanism by which mast cells induce expulsion of these parasites has not been elucidated, it has been proposed that mast cell mediators, including cytokines and granule chymases, act to create an environment inhospitable to the parasite, part of this being the induction of intestinal inflammation. Therefore, the aims of this study were to dissect the role of mast cells and mast cell proteases in the induction of parasite-induced enteropathy. METHODS: Mast cell-deficient W/Wv and mouse mast cell protease-1 (mMCP-1)-deficient mice were infected with T. spiralis, and parasite expulsion, enteropathy, and Th2 responses were determined. RESULTS: Expulsion of the parasite was delayed in both strains of mice compared with wild-type controls; additionally, in both cases, the enteropathy was significantly ameliorated. Although Th2 responses were significantly reduced in mast cell-deficient W/Wv mice, those from mMCP-1-deficient mice were similar to wild-type mice. Additionally, levels of TNF-alpha and nitric oxide were significantly reduced in both W/Wv and mMCP-1 deficient mice. CONCLUSIONS: These results imply that mast cells may contribute to the induction of protective Th2 responses and, importantly, that the intestinal inflammation associated with gastrointestinal helminths is partly mediated by mMCP-1.     

Accumulation of eosinophils in intestine-draining mesenteric lymph nodes occurs after Trichuris muris infection

Eosinophils have recently been demonstrated capable of localizing to lymph nodes that drain mucosal surfaces, in particular during T helper 2 (Th2) responses. Resistance of mice to infection with the gastrointestinal nematode Trichuris muris depends critically on mounting of a Th2 response and represents a useful model system to investigate Th2 responses. Following infection of resistant BALB/c mice with T. muris, we observed accumulation of eosinophils in intestine-draining mesenteric lymph nodes (MLNs). The accumulation of MLN eosinophils was initiated during the second week of infection and peaked during worm expulsion. In contrast, we detected a comparably late and modest increase in eosinophil numbers in the MLNs of infected susceptible AKR mice. MLN eosinophils localized preferentially to the medullary region of the lymph node, displayed an activated phenotype and contributed to the interleukin-4 (IL-4) response in the MLN. Despite this, mice genetically deficient in eosinophils efficiently generated IL-4-expressing CD4(+) T cells, produced Th2 cytokines and mediated worm expulsion during primary T. muris infection. Thus, IL-4-expressing eosinophils accumulate in MLNs of T. muris-infected BALB/c mice but are dispensable for worm expulsion and generation of Th2 responses, suggesting a distinct or subtle role of MLN eosinophils in the immune response to T. muris infection.     

Trichinella spiralis antigens prime mixed Th1/Th2 response but do not induce de novo generation of Foxp3+ T cells in vitro

Many parasitic helminth infections induce Th2-type immune responses and engage the regulatory network. In this study, we specifically investigated the influence of antigens derived from different life stages of the helminth Trichinella spiralis on the polarization of naive CD4(+) T cells by dendritic cells. Results obtained from C57BL/6 mice showed that T. spiralis derived antigens have the capacity to induce bone marrow-derived dendritic cells to acquire an incompletely mature phenotype that promotes a significant proliferation of naive CD4(+) T cells and a mixed Th1/Th2 cytokine profile with the predominance of Th2 cytokines. Increased production of IL-4, IL-9, IL-10 and IL-13 accompanied increased IFN-γ. Furthermore, dendritic cells pulsed with T. spiralis antigens did not induce an increase in the population of Foxp3(+) T regulatory cells. Although other helminth antigens have demonstrated the capacity to induce de novo generation of Foxp3(+) T regulatory cells, here our in vitro studies provide no evidence that T. spiralis antigens have this capacity.     

A comparison of local and peripheral parasite-specific antibody production in different strains of mice infected with Trichuris muris

The serum parasite-specific antibody responses of different mouse strains infected with Trichuris muris reflect the nature of the T-helper response mounted by the host, in that resistant Th2-responding strains, such as BALB/K, produce immunoglobulin (Ig)G1 and susceptible predominantly Th1-responding strains, such as AKR, produce IgG2a and IgG1. However, the kinetics of antibody production in the sera, as determined by enzyme-linked immunosorbent assay, do not reflect infection status in that resistant strains can expel their worm burdens before antibodies are detectable in the sera. Here, we show that parasite-specific antibody production by in vitro lipopolysaccharide-stimulated mesenteric lymph node cells (MLN) not only correlate with serum antibody isotypes, but also follow expulsion kinetics. Additionally, the antibody levels seen locally match changes in absolute B220+ cell numbers in the MLN (determined by flow cytometry) and changes in MLN parasite-specific plasma cells in the MLN (determined by ELISPOT). These results show that B cell responses are tightly regulated locally in both resistant and susceptible strains of mice infected with T. muris.     

Correlations between worm burden and markers of Th1 and Th2 cell subset induction in an inbred strain of mouse infected with Trichuris muris

Helper T cell subset induction was examined within a single inbred strain of mouse ( B10.D2/n) where individuals varied in their ability to expel the nematode parasite Trichuris muris. In this mouse strain approximately half of infected individuals resist infection whilst half are unable to expel the parasite and harbour chronic mature adult worm infections. We here assess various T cell and serological parameters in individual B10.D2/n mice infected with T. muris in relation to the number of parasites harboured. Worm burdens showed very significant negative correlations with five different parameters indicative of the selective expansion within the host of helper T cells of the Th2 subset. Thus, in vitro IL-5 and 1L-9 production by restimulated mesenteric lymph node cells, totallgE levels, the early parasite-specific IgG1 response (all P < 0·01) and intestinal eosinophilia (P < 0·05), were all significantly negatively correlated with worm burden. In addition, levels of IL-3 were significantly greater in mice resistant to infection (P < 0·01). In contrast there was a significant positive correlation between worm burden and parasite-specific IgG2a levels (P < 0·05), IgG2a production being under the tight control of the Th1-specific cytokine IFN-y and thus a reliable marker for in vivo Th1 cell activation. The data demonstrates that an individual infected with T. muris is capable of mounting either a protective Th2-type response or an inappropriate Th1-type response. Thus, under conditions where host genetic factors and route of antigen introduction into the host are identical, polarized helper T cell responses can arise and hence may be due to a parasite-derived influence rather than an intrinsic difference between hosts per se.     

Regulation of the immune response to intestinal nematode infections in mice

Control of parasitic infections is dependent on mechanisms that limit invasion, reproduction or survival of the parasite, including elevated serum IgE, eosinophilia and intestinal mast cell hyperplasia. Studies with mice infected with Heligmosomoides polygyrus, Trichuris muris, Nippostrongylus brasiliensis and Trichinella spiralis have provided considerable information about immune mechanisms correlated with resistance and susceptibility. Activation and cytokine secretion of distinct Th cell subset leads to the generation of effective or ineffective responses resulting in clearance of the parasite load or maintenance of chronic infection. The induction of differential responses remains to be determined but is likely to be influenced at a number of levels including the host genetic background, involvement of accessory cells, activation of co-stimulatory molecules on antigen presenting cells. The regulation of responses to intestinal nematode infections is discussed.     

Cytokine profile of protective anti-Trichinella spiralis CD4+ OX22− and non-protective CD4+ OX22+ thoracic duct cells in rats: secretion of IL-4 alone does not determine protective capacity

We analysed the cytokine profile of a T cell subset (CD4⁺ CD45 RC⁻) that confers protection against Trichinella spiralis infection in rats. These CD4⁺ cells are generated in the gut and appear in the thoracic duct lymph within 72 h after infection. Cytokine mRNA levels for IL-2, IL-3, IL-4, IL-5, IL-10 and IFN-γ and functional cytokine secretion for IL-4, IL-5, IFN-γ, TNF-α and mast cell differentiation activity were tested in vitro following stimulation with T. spiralis antigens. Compared to a non-protective T cell population (CD4⁺ CD45 RC⁺ or CD8⁺), also isolated from the same thoracic lymph, no significant differences were observed in the levels of mRNA for IL-2, IL-3, IL-4, IL-5, IL-10 or IFN-γ in the protective CD4⁺ CD45 RC⁻ cells. However, analysis of the cytokine activities in culture supernatant of these T cell subsets following 24 h stimulation in vitro with T. spiralis antigens showed that significant IL-4 and IL-5 activity but little IFN-γ or TNF-α was secreted by the protective CD4⁺ CD45 RC⁻ cells. Whereas the non-protective CD4⁺ CD45 RC⁺ cells secreted significant levels of IL-4, IFN-γ, mast cell differentiating activity and TNF-α but little IL-5 activity. Nonprotective CD8⁺ cells were found to secrete IL-4 but not IL-5. Production of IL-4 was essentially equal for both protective and non-protective T cell subsets. These findings suggest that the presence or absence of IFN-γ secretion, rather than IL-4 alone, determines whether a T cell subset has protective activity against T. spiralis infection in rats.     

环孢素A对感染旋毛虫小鼠的免疫调节作用

目的观察环孢素A(CsA)对感染旋毛虫小鼠免疫功能的影响。方法小鼠腹腔注射环孢素A感染旋毛虫后,分别采用ELISA和流式细胞仪检测小鼠不同时期外周血中IL-2含量、CD4+及CD8+T淋巴细胞的百分率。结果(1)单纯感染旋毛虫小鼠感染后1~5周IL-2的含量较正常组明显增加;注射CsA组小鼠感染后1周IL-2的含量较单纯感染组明显降低,感染后2~4周IL-2的含量与单纯感染组相比无明显差异,但仍高于正常组。(2)单纯感染旋毛虫小鼠感染后1~4周,CD4+T淋巴细胞较正常组明显减少,CD8+T淋巴细胞明显增多,CD4+/CD8+比值明显下降;注射CsA组小鼠CD4+T淋巴细胞细胞较单纯感染组明显减少,而CD8+T淋巴细胞无明显变化。结论CsA对感染旋毛虫小鼠具有一定的免疫调节作用。     

The role of IL-4, IL-13 and IL-4Ralpha in the development of protective and pathological responses to Trichinella spiralis

T helper type 2 (Th2) responses have been shown to be important in protective responses to gastrointestinal (GI) helminth infections and in the development of the intestinal pathology accompanying expulsion of the parasite. Different inbred mouse strains have been shown to develop different cytokine profiles following infection with GI helminths with increased resistance observed in those strains where Th2 cytokines predominate. The aim of this study was to determine the role of IL-4, IL-13 and IL-4Ralpha and the impact of host background on the development of the protective and pathological responses induced by infection with the gastrointestinal helminth Trichinella spiralis. IL-4, IL-13 and IL-4Ralpha were required for the generation of Th2 responses to T. spiralis; however, the role these responses play in the development of protection and enteropathy was less clear. IL-4Ralpha-deficiency mice resulted in substantially reduced parasite expulsion, intestinal pathology and Th2 responses. Similarly, lack of IL-13 resulted in inhibited expulsion and the development of enteropathy. Although Th2 responses were reduced in BALB/c IL-4-/- mice, neither expulsion nor enteropathy were different from wild-type mice. In contrast, C57BL/6 IL-4-/- exhibited delayed expulsion and reduced pathology, suggesting that host genetics are important in the function of individual cytokines. Thus, differences in background genotype may be an important component in the development host protection and the development of intestinal pathology accompanying the loss of GI helminths.     

IL-33: a Janus cytokine

Interleukin (IL) 33, a member of the IL-1 family, is the ligand of ST2 that is expressed mainly on activated Th2 cells and mast cells. IL-33 can skew a predominantly Th1 cell population to a mainly Th2 cells phenotype in vivo. IL-33 messenger RNA is expressed early during infection of the intestinal-dwelling nematode Trichuris muris in mice. IL-33 treatment enhances resistance to Trichuris infection. IL-33 also effectively attenuates sepsis by mobilising the innate cells, neutrophils, to the site of infection, helping to clear the pathogens. Thus, IL-33 may be evolutionally preserved for the host defence against infections. IL-33 can reduce an ongoing atherosclerosis in ApoE(-/-) mice and attenuate adipocytes mainly by inducing the production of type II cytokines. In contrast, IL-33 can also exacerbate allergy and the inflammation in collagen-induced or serum-induced arthritis. Hence, IL-33 is a double-edged sword, and targeting IL-33 should be approached with caution.     

Suppression of muscosal mastocytosis by infection with the intestinal nematode Nematospiroides dubius

Mice exposed to primary infections with the parasite intestinal nematode Nematospiroides dubius failed to show the mucosal mast cell (MMC) response which is characteristic of infections with other species of intestinal nematode and which was readily induced in these mice by infections with Nippostrongylus brasiliensis or Trichinella spiralis. The failure to generate a mucosal mastocytosis was independent of host strain or sex. When infections with N. dubius were established before, or concurrently with, T. spiralis or N. brasiliensis, the MMC response elicited by these species was delayed and/or depressed as was expulsion of the worms themselves. Infection with N. dubius given when a MMC response was already established, by exposure to T. spiralis, had no effect on MMC numbers. The possibility that the effects of N. dubius upon MMC responses reflect a lack of mastocytopoietic potential, rather than an active interference, was excluded by showing that SJL mice, which expel primary infections with N. dubius and express strong immunity to reinfection, developed marked mastocytosis during secondary infections. The depression of MMC responses by N. dubius is discussed in relation to the known immunosuppressive properties of this parasite and in relation to the T cell mediated control of MMC development.     

Characterization of rat bone marrow dendritic cells initially primed by Trichinella spiralis antigens

Pathogen-derived products have the capacity to induce maturation of bone marrow-derived dendritic cells (BMDCs) into populations of effectors cells that polarize Th cells toward Th1 or Th2 phenotype via different mechanisms. Since those mechanisms are not entirely clear for helminths, and almost completely unknown for Trichinella spiralis (TS), we started an investigation of the effects of TS antigens (four different antigens isolated from all three life-cycle stages of parasite) on maturation of BMDCs and their potential to present TS antigens. The expression of MHC class II, costimulatory molecules CD86, CD54, IL-10 and IL-12p70 cytokine production were measured after 2 days of BMDCs cultivation with TS antigens. While parasitic antigens did not significantly alter the expression of MHC II, most of them, except crude muscle larvae antigens, up-regulated the expression of costimulatory molecules. BMDCs, primed with all TS antigens, released increased amounts of IL-10 and decreased amounts of IL-12 p70. BMDCs, primed with TS antigens, induced significant proliferation of syngeneic TS sensitized lymph nodes cells and also stimulated the production of IL-4 by T cells purified from of TS infected DA rats. The results indicate that TS stimulated BMDCs leads to the polarization of the immune response towards regulatory and Th2 type.