Lactic dehydrogenase virus infection enhances parasite egg production and inhibits eosinophil and mast cell responses in mice infected with the nematode Nippostrongylus brasiliensis.

The effects of lactic dehydrogenase virus (LDV) infection on the protective immune responses to the nematode Nippostrongylus brasiliensis were studied. Mice with chronic LDV infection showed significantly higher levels of parasite egg production than non-LDV-infected (control) mice after N. brasiliensis infection. Concurrent LDV infection also suppressed peripheral blood eosinophilia and the lung mastocytosis induced by this nematode. LDV infection showed higher expression levels of the interferon-gamma (IFN-gamma) mRNA in lymph nodes compared with control mice before N. brasiliensis infection. In addition, the IgG2a production in LDV-infected mice was higher than that in control mice before and after N. brasiliensis infection. These results suggest that LDV infection modulates protective immune responses against N. brasiliensis infection by the activation of T-helper type 1 cells.     

Induction of the expulsion of Strongyloides ratti and retention of Nippostrongylus brasiliensis in athymic nude mice by repetitive administration of recombinant interleukin-3.

A repetitive administration of recombinant interleukin-3 (rIL-3), which can induce the expulsion of Strongyloides ratti in athymic nude mice, did not affect the expulsion of Nippostrongylus brasiliensis. Nude mice infected with N. brasiliensis were injected i.p. with a total of 6.8 x 10(5) U rIL-3 or medium twice a day from Day 5 to Day 11 post-infection. The kinetics of expulsion estimated by egg excretion in faeces up to Day 20 post-infection and adult worm burden on Day 21 was not affected by the IL-3 administration. A similar administration with a higher dose (total 10.6 x 10(5) U) of rIL-3 did not alter the adult worm burden on Day 13. The number of intestinal mucosal mast cells on Day 13 was markedly increased by the treatment, although the number of intestinal goblet cells was comparable between the treated and control mice. When nude mice were infected concurrently with N. brasiliensis and S. ratti and then injected repeatedly with rIL-3 (total 2.2 x 10(5) U) from Day 5 to Day 11, adult worms of S. ratti were expelled from the small intestine by Day 13; however adult worms of N. brasiliensis were retained. Again in the concurrent infection, the number of intestinal mucosal mast cells was significantly increased but that of intestinal goblet cells was not altered by the rIL-3 administration. These results indicate that the expulsion of S. ratti is dependent on IL-3 whereas that of N. brasiliensis is less dependent on IL-3.     

Regulation of immune complexes during infection of mice with lactate dehydrogenase-elevating virus: studies with interferon-gamma gene knockout and tolerant mice.

Mice persistently infected with lactate dehydrogenase-elevating virus (LDV) develop circulating IgG-containing hydrophobic immune complexes, with a molecular mass of 150 to 300 kd, which bind to the surfaces of high-capacity enzyme-linked immunosorbent assay (ELISA) plates. LDV infection also stimulates polyclonal B-cell activation and autoimmunity. For this study, interferon-gamma gene knockout (GKO) mice were utilized to study circulating immune complexes and other parameters of LDV infection. The kinetics of LDV viremia, formation of plasma IgG anti-LDV antibodies, and LDV replication in the spleen and liver were essentially normal in GKO mice. Polyclonal activation of B cells, as reflected by increased total plasma IgG concentration during LDV infection, was found to be intact in GKO mice, although at a lower magnitude than in control mice. The plasma concentration of IgG-containing hydrophobic immune complexes was reduced about 75% in LDV-infected GKO mice relative to normal LDV-infected controls. Allogeneic tissue responses were also found to be reduced in LDV-infected GKO mice relative to those in normal LDV-infected controls. These results dissociate specific anti-LDV immunity from formation of hydrophobic immune complexes, show that the IgG anti-LDV response as well as LDV replication in the spleen and liver are insensitive to physiological levels of interferon (IFN)-gamma, and suggest that IgG-containing immune complexes stimulated by LDV infection are a marker for autoimmunity.     

Precision and linearity targets for validation of an IFNγ ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides

Background

Intestinal mucus production by hyperplasic goblet cells is a striking pathological feature of many parasitic helminth infections and is related to intestinal protection and worm expulsion. Induction of goblet cell hyperplasia is associated with TH2 immune responses, which in helminth infections are controlled primarily by IL-13, and also IL-4. In the study presented here we examine the goblet cell hyperplasic response to three experimental parasitic helminth infections; namelyNippostrongylus brasiliensis,Syphacia obvelataandSchistosoma mansoni.

Results

As expectedN. brasiliensisinfection induced a strong goblet cell hyperplasia dependent on IL-4/IL-13/IL-4Rα expression. In contrast, and despite previously published transiently elevated IL-4/IL-13 levels,S. obvelatainfections did not increase goblet cell hyperplasia in the colon. Furthermore, induction of goblet cell hyperplasia in response toS. mansonieggs traversing the intestine was equivalent between BALB/c, IL-4/IL-13^-/-and IL-4Rα^-/-mice.

Conclusion

Together these data demonstrate that intestinal goblet cell hyperplasia can be independent of TH2 immune responses associated with parasitic helminth infections.     

Expulsion of Nippostrongylus brasiliensis from mice lacking antibody production potential.

Expulsion of the intestinal nematode of rodents, Nippostrongylus brasiliensis, was assessed in mice experiencing the immunosuppressive effects of anti-micron antibodies. Anti-micron treatment resulted in complete elimination of IgM and severe reduction of IgG1, IgG2 and IgA serum immunoglobulin levels. Specific antibody responses to sheep erythrocytes were virtually eliminated in anti-micron-treated mice as determined by direct and indirect plaque-forming-cell responses, haemagglutination and haemolytic assays. Using passive cutaneous anaphylaxis (PCA) and indirect haemagglutination (IHA) assays, antibodies against N. brasiliensis were not detectable in sera of anti-micron-treated mice; control mice, however, generated strong PCA and IHA responses. The kinetics of worm egg production coupled with adult worm recoveries at necropsy indicate that anti-micron treatment of mice had little or no effect on the capacity of mice to expel N. brasiliensis even though antibody production potential had been eliminated in these mice. Thus, anti-worm antibodies may not be requisite in the mechanism of N. brasiliensis expulsion from mice.     

Lactic Dehydrogenase Virus Inhibits Allergic Immunoglobulin E Production: In Vivo Molecular Analysis of Cytokines

The effects of lactic dehydrogenase virus (LDV) infection on allergic immunoglobulin (Ig)E production and interleukin (IL)-4 gene expression were studied. LDV infection suppressed antigen-induced IgE production in sensitized mice. The elevations of IL-4 gene expression in spleen and mesenteric lymph nodes 3 and 7 days after ovalbumin challenge were suppressed significantly in LDV-infected mice compared with control mice. The expression of the interferon (IFN)-gamma gene of mesenteric lymph nodes was significantly increased in LDV-infected mice. These results suggest that LDV infection suppressed antigen-induced IgE production by decreasing IL-4 production, and that suppression of IL-4 gene expression may be mediated by a mutual inhibition mechanism between T helper (Th)1 and Th2 cells.     

Lactobacillus rhamnosus Ingestion Promotes Innate Host Defense in an Enteric Parasitic Infection

Enteric parasite infections around the world are a huge economic burden and decrease the quality of life for many people. The use of beneficial bacteria has attracted attention for their potential therapeutic applications in various diseases. However, the effects of beneficial bacteria in enteric parasitic infections remain largely unexplored. We investigated the effects of ingestion of Lactobacillus rhamnosus (JB-1) in a model of enteric nematode (Trichuris muris) infection. C57BL/6 (resistant to infection), AKR (susceptible to infection), interleukin 10 (IL-10) knockout (KO), and mucin Muc2 KO mice were infected with T. muris and treated orally with probiotic JB-1 or medium. The mice were sacrificed on various days postinfection to examine goblet cells, epithelial cell proliferation, cytokines, and worm burdens. Treatment with JB-1 significantly enhanced worm expulsion in resistant C57BL/6 mice, and this was associated with increases in IL-10 levels, goblet cell numbers, and epithelial cell proliferation. Beneficial effects of JB-1 were absent in IL-10 KO and resistant mice treated with γ-irradiated bacteria. Live JB-1 treatment also expedited worm expulsion in Muc2 KO mice and, more importantly, in AKR mice (susceptible to infection). Injection of IL-10 directly into the colonic tissue of uninfected mice induced goblet cell hyperplasia. These findings demonstrate that JB-1 modulates goblet cell biology and promotes parasite expulsion via an IL-10-mediated pathway and provide novel insights into probiotic effects on innate defense in nematode infection.     

γδ T cells play a protective role during infection with Nippostrongylus brasiliensis by promoting goblet cell function in the small intestine

The intestinal epithelium is rich in γδ T cells and the gut is a site of residence for a wide variety of pathogens, including nematodes. Although CD4+ T-cell receptor (TCR) -αβ+ T helper type 2 T cells are essential for the expulsion of intestinal nematodes, little information is available on the function of γδ T cells in this type of infection. Here, we demonstrate two major functions of γδ T cells as a potently protective T-cell population against Nippostrongylus brasiliensis infection using γδ T-cell-deficient (TCR-δ(-/-) ) mice. First, γδ T cells are required to initiate rapid expulsion of adult worms from the intestine and to limit egg production. Second, γδ T cells prevent the pathological intestinal damage associated with nematode infection, evident by increased clinical disease and more severe microscopic lesions in infected TCR-δ(-/-) mice. γδ T-cell deficiency led to delayed goblet cell hyperplasia in association with reduced expression of phosphorylated STAT6, MUC2, Trefoil factor-3 (TFF3) and T helper type 2 cytokines including interleukin-13 (IL-13). TCR-δ(-/-) mice also produced more interferon-γ than wild-type mice. Within the intraepithelial lymphocyte compartment, γδ T cells produced IL-13. Adoptive transfer of γδ T cells or administration of recombinant IL-13 to TCR-δ(-/-) mice successfully reduced the egg production by N. brasiliensis. Collectively, these data provide strong evidence that γδ T cells play an important role in controlling infection with intestinal nematodes and limiting infection-induced pathology.     

Infection with Toxoplasma gondii reduces established and developing Th2 responses induced by Nippostrongylus brasiliensis infection

Oral infection of C57BL/6 mice with 100 cysts of the protozoan parasite Toxoplasma gondii results in the development of small intestinal Th1-type immunopathology. In contrast, infection with intestinal helminths results in the development of protective Th2-type responses. We investigated whether infection with the helminth Nippostrongylus brasiliensis influences the development of T. gondii-induced Th1 responses and immunopathology in C57BL/6 mice infected with T. gondii. Prior as well as simultaneous infection of mice with N. brasiliensis did not alter the course of infection with 100 cysts of T. gondii. Coinfected mice produced high levels of interleukin-12 (IL-12) and gamma interferon (IFN-gamma), developed small intestinal immunopathology, and died at the same time as mice infected with T. gondii. Interestingly, local and systemic N. brasiliensis-induced Th2 responses, including IL-4 and IL-5 production by mesenteric lymph node and spleen cells and numbers of intestinal goblet cells and blood eosinophils, were markedly lower in coinfected than in N. brasiliensis-infected mice. Similar effects were seen when infection with 10 T. gondii cysts was administered following infection with N. brasiliensis. Infection of C57BL/6 mice with 10 T. gondii cysts prior to coinfection with N. brasiliensis inhibited the development of helminth-induced Th2 responses and was associated with higher and prolonged N. brasiliensis egg production. In contrast, oral administration of Toxoplasma lysate prior to N. brasiliensis infection had only a minor and short-lived effect on Th2 responses. Thus, N. brasiliensis-induced Th2 responses fail to alter T. gondii-induced Th1 responses and immunopathology, most likely because Th1 responses develop unchanged in C57BL/6 mice with a prior or simultaneous infection with N. brasiliensis. Our findings contribute to the understanding of immune regulation in coinfected animals and may assist in the design of immunotherapies for human Th1 and Th2 disorders.     

Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology

Macrophage/neutrophil-specific IL-4 receptor alpha-deficient mice (LysM(Cre)IL-4Ralpha(-/flox)) were generated to understand the role of IL-4/IL-13 responsive myeloid cells during Type 2 immune responses. LysM(Cre)IL-4Ralpha(-/flox) mice developed protective immunity against Nippostrongylus brasiliensis accompanied by T(H)2 development and goblet cell hyperplasia. In contrast, LysM(Cre)IL-4Ralpha(-/flox) mice were extremely susceptible to Schistosoma mansoni infection with 100% mortality during acute infection. Mortality was not dependent on neutrophils and occurred in the presence of T(H)2/Type 2 responses, granuloma formation, and egg-induced fibrosis. Death was associated with increased T(H)1 cytokines, hepatic and intestinal histopathology, increased NOS-2 activity, impaired egg expulsion, and sepsis. IL-10 was not able to compensate for the absence of IL-4/IL-13-activated alternative macrophages. Together, this shows that alternative macrophages are essential during schistosomiasis for protection against organ injury through downregulation of egg-induced inflammation.     

Lactic dehydrogenase virus infection inhibits allergic eosinophil reaction and IL-5 gene expression in vivo.

The effects of lactic dehydrogenase virus (LDV) infection on allergic eosinophil reaction and IL-5 gene expression were studied. LDV infection suppressed antigen-induced eosinophil recruitment into the peritoneal cavity in sensitized mice. The elevation of IL-5 gene expression in the spleen and mesenteric lymph nodes 6 h after ovalbumin challenge was significantly suppressed in LDV-infected mice compared with uninfected (control) mice. The expression of the interferon-gamma and IL-2 genes in the spleen, but not in mesenteric lymph nodes, was significantly suppressed in LDV-infected mice compared with control mice. The present results suggest, that suppression of IL-5 gene expression by LDV infection may not be mediated by a mutual inhibitory mechanism between Th1 and Th2 cells.     

Immune responses to Nippostrongylus brasiliensis and tuberculin protein in GATA-3-transgenic mice

GATA-3 appears to be key to the Th2 response. However, few in vivo experiments have examined the function of GATA-3 in Th1 and Th2 immune responses. We developed two lines of GATA-3-transgenic (Tg) mice harboring the SRalpha or lck promoters and examined the Th2 immune responses of mice infected with the intestinal nematode Nippostrongylus brasiliensis and the Th1 responses with purified derivative of tuberculin (PPD) immunization. Numbers of peripheral blood eosinophils in all GATA-3-Tg mice increased 10- to 20-fold after primary infection with N. brasiliensis and 25-100-fold after secondary infection. The number of eosinophils in infected GATA-3-Tg mice was significantly higher than that in infected control littermates. Total IgE levels after primary infection in GATA-3-Tg mice were 8-450-fold increased, which was significantly higher than those of control mice. Mesenteric lymph node cells of infected GATA-3-Tg mice upon stimulation with N. brasiliensis antigen secreted more IL-5 and IL-13 than those of control mice. However, production of IL-4 and IFN-gamma were comparable between GATA-3-Tg and controls. Mice immunized with PPD were intradermally challenged with PPD to induce delayed type hypersensitivity (DTH). The amount of footpad swelling caused by the DTH reaction in GATA-3-Tg mice was significantly smaller than that of control littermates. Inguinal lymph node cells from GATA-3-Tg mice stimulated with PPD in vitro secreted more IL-5, IL-10 and less IFN-gamma than those of control littermates. These results suggested that Th1 and Th2 driven conditions enhance IL-5 production in GATA-3-Tg mice through the direct binding of GATA-3 to the IL-5 promoter region. The influence of GATA-3 on IL-13, IFN-gamma and IL-10 production varied according to the stimulating conditions. However, IL-4 production was not significantly elevated in GATA-3-Tg mice, indicating that IL-4 and IL-5 production was differentially regulated in these mice.     

Conditional IL-4/IL-13-deficient mice reveal a critical role of innate immune cells for protective immunity against gastrointestinal helminths

Approximately one-third of the world population is infected with gastrointestinal helminths. Studies in mouse models have demonstrated that the cytokines interleukin (IL)-4 and IL-13 are essential for worm expulsion, but the critical cellular source of these cytokines is poorly defined. Here, we compared the immune response to Nippostrongylus brasiliensis in wild-type, T cell-specific IL-4/IL-13-deficient and general IL-4/IL-13-deficient mice. We show that T cell-derived IL-4/IL-13 promoted T helper 2 (Th2) polarization in a paracrine manner, differentiation of alternatively activated macrophages, and tissue recruitment of innate effector cells. However, innate IL-4/IL-13 played the critical role for induction of goblet cell hyperplasia and secretion of effector molecules like Mucin5ac and RELMβ in the small intestine. Surprisingly, T cell-specific IL-4/IL-13-deficient and wild-type mice cleared the parasite with comparable efficiency, whereas IL-4/IL-13-deficient mice showed impaired expulsion. These findings demonstrate that IL-4/IL-13 produced by cells of the innate immune system is required and sufficient to initiate effective type 2 immune responses resulting in protective immunity against N. brasiliensis.     

The requirement of thymus competence for both humoral and cell-mediated steps in expulsion of Nippostorngylus brasiliensis from mice.

The worm damaging process (step 1 of Nippostrongylus brasiliensis expulsion from rodents) does not occur in congenitally thymus-deficient (nude) mice as determined by worm morphology, female worm fecundity, and kinetics of worm expulsion upon transfer into normal rats. Expulsion of damaged N. brasiliensis (step 2) does not occur subsequent to transfer of such worms from rats into nude mice. Morphological changes and reduced fecundity appeared in adult worms from normal thymus-bearing mice as early as the first day of patency (day 6 of infection). Thus, the worm damaging process is initiated several days earlier in mice than in rats and may, therefore, account for the reduced longevity of N. brasiliensis in mice compared with rats.     

Involvement of ICOS-B7RP-1 costimulatory pathway in the regulation of immune responses to Leishmania major and Nippostrongylus brasiliensis infections

The ICOS-B7RP-1-mediated T cell costimulatory pathway has been implicated crucial for T cell activation and differentiation. In this study, we investigated the role of this costimulation in the regulation of immune responses to parasitic infections by using blocking antibody against B7RP-1 as well as ICOS-deficient mice. The administration of anti-B7RP-1 monoclonal antibody (mAb) significantly suppressed the footpad swelling in susceptible BALB/c mice upon Leishmania major infection. The observation was consistent not only with the significant suppression of IL-4, IL-5 and IL-10 secretion from lymph node cells, which were derived from L. major-infected mice, but also with the significant reduction of total serum IgE and IgG(1) in anti-B7RP-1 mAb-treated BALB/c mice. Infection of ICOS-deficient mice with L. major also suggested the impaired Th2 immune responses in the absence of this costimulation. The immunological function of ICOS-B7RP-1 costimulatory pathway in infection was further confirmed by infecting anti-B7RP-1 mAb-treated wild type or ICOS-deficient mice with Nippostrongylus brasiliensis. The characteristic elevation of total serum IgE and eosinophilia upon N. brasiliensis infection was suppressed by blocking this costimulation. Moreover, the protection to N. brasiliensis adult worms was suppressed in anti-B7RP-1 mAb-treated wild type or ICOS-deficient mice. These results suggest the crucial role of this costimulatory pathway in the regulation of Th2-biased T cell differentiation and in host immune responses against L. major and N. brasiliensis infections.     

IL-9-deficient mice establish fundamental roles for IL-9 in pulmonary mastocytosis and goblet cell hyperplasia but not T cell development

Interleukin-9 is a cytokine produced by Th2 cells and is a candidate gene for asthma and atopy. We have generated IL-9-deficient mice to delineate the specific roles of IL-9 in Th2 responses. Using a pulmonary granuloma model, we have demonstrated a distinct requirement for IL-9 in the rapid and robust generation of pulmonary goblet cell hyperplasia and mastocytosis in response to lung challenge. In contrast, eosinophilia and granuloma formation were not affected. IL-9 was not required for T cell development or differentiation, the generation of naive or antigen-driven antibody responses, or the expulsion of the intestinal parasitic nematode Nippostrongylus brasiliensis. Thus, deletion of IL-9 manifests as a highly defined phenotype in Th2 responses modulating mucus production and mast cell proliferation.     

Interleukin-13 Receptor α1-Dependent Responses in the Intestine Are Critical to Parasite Clearance

Nematode infection upregulates interleukin-4 (IL-4) and IL-13 and induces STAT6-dependent changes in gut function that promote worm clearance. IL-4 and IL-13 activate the type 2 IL-4 receptor (IL-4R), which contains the IL-13Rα1 and IL-4Rα chains. We used mice deficient in IL-13Rα1 (IL-13Rα1^−/−) to examine the contribution of IL-13 acting at the type 2 IL-4R to immune and functional responses to primary (Hb1) and secondary (Hb2) infections with the gastrointestinal nematode parasite Heligmosomoides bakeri. There were differences between strains in the IL-4 and IL-13 expression responses to Hb1 but not Hb2 infection. Following Hb2 infection, deficient mice had impaired worm expulsion and higher worm fecundity despite normal production of Th2-derived cytokines. The upregulation of IL-25 and IL-13Rα2 in Hb1- and Hb2-infected wild-type (WT) mice was absent in IL-13Rα1^−/− mice. Goblet cell numbers and resistin-like molecule beta (RELM-β) expression were attenuated significantly in IL-13Rα1^−/− mice following Hb2 infections. IL-13Rα1 contributes to the development of alternatively activated macrophages, but the type 1 IL-4R is also important. Hb1 infection had no effects on smooth muscle function or epithelial permeability in either strain, while the enhanced mucosal permeability and changes in smooth muscle function and morphology observed in response to Hb2 infection in WT mice were absent in IL-13Rα1^−/− mice. Notably, the contribution of claudin-2, which has been linked to IL-13, does not mediate the increased mucosal permeability following Hb2 infection. These results show that activation of IL-13Rα1 is critical for key aspects of the immune and functional responses to Hb2 infection that facilitate expulsion.     

Immune-mediated alteration of the terminal sugars of goblet cell mucins in the small intestine of Nippostrongylus brasiliensis-infected rats.

Alteration of terminal sugars of goblet cell mucins in the small intestinal mucosa was examined by lectin histochemistry in rats infected with Nippostrongylus brasiliensis. To see whether alteration of the nature of mucins was regulated by the local immune system, adult worms were implanted intraduodenally into recipient rats of various immune status. When 7-day-old ('normal') adult worms were implanted into naive euthymic rats, about 60% remained until day 7 and then were expelled. The number of goblet cells transiently decreased on day 5 and then progressively increased from day 9 onwards. In parallel with the hyperplasia, mucins in and released from goblet cells strongly expressed terminal N-acetyl-D-galactosamine which was specifically recognized by Helix pomatia agglutinin (HPA). When 13-day-old ('damaged') adult worms were implanted into naive euthymic rats, they were rapidly expelled by day 9 in association with hastened goblet cell hyperplasia and the alteration of terminal sugars of the mucins. Hastened worm expulsion, goblet cell hyperplasia and alteration of terminal sugars of the mucins were also observed when 'normal' worms were implanted into immune recipients. On the other hand, after implantation of 'normal' worms into hypothymic (rnu/rnu) rats, goblet cell hyperplasia or the alteration of terminal sugars of the mucins was almost completely absent. These results suggest that alteration of sugar residues of goblet cell mucins, especially the strong expression of terminal N-acetyl-D-galactosamine, is regulated by the host's immune system and seems to be important in the expulsion of N. brasiliensis.     

The effectors responsible for gastrointestinal nematode parasites, Trichinella spiralis, expulsion in rats

Helper T (Th2) cells type 2 have a central role in host protective responses to gastrointestinal nematode parasite, Trichinella spiralis infection, but the actual effector mechanisms involved in parasite expulsion are still uncertain. Recent evidences suggest that mast cell recruitment and activation may associate with parasite elimination from host intestines in mice. On the other hand, IgE production may induce defensive responses to primary infection with the helminth in rats. The differences of host effector mechanisms to the same experimental infections might disturb our understanding of the host protective mechanisms to gastrointestinal nematode parasite infection. In order to redefine these differences, we investigated in detail the relationship between intestinal immune responses and worm expulsion following T. spiralis infection among several rat strains including mutants. As a result, there were significant correlations of parasite expulsion with mast cell hyperplasia in addition to serum IgE level. Moreover, mast cell-deficient and dysfunction rats showed delayed worm elimination from their gut. Therefore, the present study suggests that mast cells should also be one of the prominent effector cells involved in T. spiralis parasite expulsion in rats as well as mice.