Sigmodontis Infection Research Articles

Th2 Cell-Intrinsic Hypo-Responsiveness Determines Susceptibility to Helminth Infection

The suppression of protective Type 2 immunity is a principal factor driving the chronicity of helminth infections, and has been attributed to a range of Th2 cell-extrinsic immune-regulators. However, the intrinsic fate of parasite-specific Th2 cells within a chronic immune down-regulatory environment, and the resultant impact such fate changes may have on host resistance is unknown. We used IL-4gfp reporter mice to demonstrate that during chronic helminth infection with the filarial nematode Litomosoides sigmodontis, CD4+ Th2 cells are conditioned towards an intrinsically hypo-responsive phenotype, characterised by a loss of functional ability to proliferate and produce the cytokines IL-4, IL-5 and IL-2. Th2 cell hypo-responsiveness was a key element determining susceptibility to L. sigmodontis infection, and could be reversed in vivo by blockade of PD-1 resulting in long-term recovery of Th2 cell functional quality and enhanced resistance. Contrasting with T cell dysfunction in Type 1 settings, the control of Th2 cell hypo-responsiveness by PD-1 was mediated through PD-L2, and not PD-L1. Thus, intrinsic changes in Th2 cell quality leading to a functionally hypo-responsive phenotype play a key role in determining susceptibility to filarial infection, and the therapeutic manipulation of Th2 cell-intrinsic quality provides a potential avenue for promoting resistance to helminths.

Chronic Helminth Infection Does Not Exacerbate Mycobacterium tuberculosis Infection

BackgroundChronic helminth infections induce a Th2 immune shift and establish an immunoregulatory milieu. As both of these responses can suppress Th1 immunity, which is necessary for control of Mycobacterium tuberculosis (MTB) infection, we hypothesized that chronic helminth infections may exacerbate the course of MTB.Methodology/Principal FindingsCo-infection studies were conducted in cotton rats as they are the natural host for the filarial nematode Litomosoides sigmodontis and are an excellent model for human MTB. Immunogical responses, histological studies, and quantitative mycobacterial cultures were assessed two months after MTB challenge in cotton rats with and without chronic L. sigmodontis infection. Spleen cell proliferation and interferon gamma production in response to purified protein derivative were similar between co-infected and MTB-only infected animals. In contrast to our hypothesis, MTB loads and occurrence and size of lung granulomas were not increased in co-infected animals.Conclusions/SignificanceThese findings suggest that chronic filaria infections do not exacerbate MTB infection in the cotton rat model. While these results suggest that filaria eradication programs may not facilitate MTB control, they indicate that it may be possible to develop worm-derived therapies for autoimmune diseases that do not substantially increase the risk for infections.

Helminth Protection against Autoimmune Diabetes in Nonobese Diabetic Mice Is Independent of a Type 2 Immune Shift and Requires TGF-β

Leading hypotheses to explain helminth-mediated protection against autoimmunity postulate that type 2 or regulatory immune responses induced by helminth infections in the host limit pathogenic Th1-driven autoimmune responses. We tested these hypotheses by investigating whether infection with the filarial nematode Litomosoides sigmodontis prevents diabetes onset in IL-4-deficient NOD mice and whether depletion or absence of regulatory T cells, IL-10, or TGF-β alters helminth-mediated protection. In contrast to IL-4-competent NOD mice, IL-4-deficient NOD mice failed to develop a type 2 shift in either cytokine or Ab production during L. sigmodontis infection. Despite the absence of a type 2 immune shift, infection of IL-4-deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4-deficient NOD mice were accompanied by increases in CD4(+)CD25(+)Foxp3(+) regulatory T cell frequencies and numbers, respectively, and helminth infection increased the proliferation of CD4(+)Foxp3(+) cells. However, depletion of CD25(+) cells in NOD mice or Foxp3(+) T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGF-β, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. Changes in Th17 responses did not seem to play an important role in helminth-mediated protection against autoimmunity, because helminth infection was not associated with a decreased Th17 immune response. This study demonstrates that L. sigmodontis-mediated protection against diabetes in NOD mice is not dependent on the induction of a type 2 immune shift but does require TGF-β.

Th2 Responses to Helminth Parasites Can Be Therapeutically Enhanced by, but Are Not Dependent upon, GITR–GITR Ligand Costimulation In Vivo

The immune suppression that characterizes human helminth infections can hinder the development of protective immunity or help to reduce pathogenic inflammation. Signaling through the T cell costimulator glucocorticoid-induced TNFR-related protein (GITR) counteracts immune downregulation by augmenting effector T cell responses and abrogating suppression by Foxp3(+) regulatory T cells. Thus, superphysiological Ab-mediated GITR costimulation represents a novel therapy for promoting protective immunity toward parasitic helminths, whereas blocking physiological GITR-GITR ligand (GITRL) interactions may provide a mechanism for dampening pathogenic Th2 inflammation. We investigated the superphysiological and physiological roles of the GITR-GITRL pathway in the development of protective and pathogenic Th2 responses in murine infection models of filariasis (Litomosoides sigmodontis) and schistosomiasis (Schistosoma mansoni). Providing superphysiological GITR costimulation using an agonistic anti-GITR mAb over the first 12 d of L. sigmodontis infection initially increased the quantity of Th2 cells, as well as their ability to produce Th2 cytokines. However, as infection progressed, the Th2 responses reverted to normal infection levels, and parasite killing remained unaffected. Despite the Th2-promoting role of superphysiological GITR costimulation, Ab-mediated blockade of the GITR-GITRL pathway did not affect Th2 cell priming or maintenance during L. sigmodontis infection. Blockade of GITR-GITRL interactions during the acute egg phase of S. mansoni infection resulted in reduced Th2 responses, but this effect was confined to the spleen and did not lead to changes in liver pathology. Thus, although superphysiological GITR costimulation can therapeutically enhance Th2 responses, physiological GITR-GITRL interactions are not required for the development of Th2-mediated resistance or pathology in murine models of filariasis and schistosomiasis.

Immunity to Parasites

Parasites such as protozoa or helminths currently account for greater morbidity and mortality than any other class of infectious organisms, particularly in developing countries. The structural and antigenic diversity of pathogenic parasites is reflected in the heterogeneity of the adaptive immune responses that they elicit. Protozoa that live within host cells are destroyed by cell-mediated immunity, whereas helminths are eliminated by IgE antibody and eosinophil mediated killing as well as by other leukocytes. The principal innate immune response to protozoa is phagocytosis, but many of these parasites are resistant to phagocytic killing and may even replicate within macrophages. Phagocytes also attack helminthic parasites and secrete microbicidal substances to kill organisms that are too large to be phagocytosed. Some helminths may also activate the alternative pathway of complement. The principal defense mechanism against protozoa that survive within macrophages (e.g. Leishmania spp., Toxoplasma gondii) is cell-mediated immunity, particularly macrophage activation by TH1 cell-derived cytokines. Protozoa that replicate inside various host cells and lyse these cells stimulate specific antibody and cytotoxic T lymphocytes (CTL) responses (e.g. Plasmodium spp.). Defense against many helminthic infections is mediated by the activation of TH2 cells, which results in production of IgE antibodies and activation of eosinophils and mast cells. The combined actions of mast cells and eosinophils lead to expulsion and destruction of the parasites. Most parasitic infections are chronic because of weak innate immunity and the ability of parasites to evade or resist elimination by adaptive immune responses Parasites evade the immune system by varying their antigens during residence in vertebrate hosts, by acquiring resistance to immune effector mechanisms, and by masking and shedding their surface antigens. Keywords: Protozoa, helminths, innate, Th1/Th2, Immunity, Parasites, adaptive immune responses, IgE, eosinophil-mediated, cell-mediated immunity, macrophages, leukocytes, Leishmania spp, Toxoplasma, Plasmodium, cytotoxic T lymphocytes, eosinophils, mast cells, immigration, immunoparasitology, immunology, Taenia solium eggs, mannose-rich lipophosphoglycan, Schistosoma mansoni, C-reactive protein, glycophosphatidylinositol, TNF, IL-1, CD80, CD86, OX40 receptor, Gata3 expression, IGF, NKT Cells, Marginal Zone, basophils, Eosinophil, caM, L-arginine hydrolysis, Litomosoides sigmodontis infection, trypomastigote, CD59, KDal-protein, SCIP-1, apolipoprotein, haptoglobin-related protein, lipophosphoglycan, E. histolytica

Basophils Amplify Type 2 Immune Responses, but Do Not Serve a Protective Role, during Chronic Infection of Mice with the Filarial Nematode Litomosoides sigmodontis

Chronic helminth infections induce a type 2 immune response characterized by eosinophilia, high levels of IgE, and increased T cell production of type 2 cytokines. Because basophils have been shown to be substantial contributors of IL-4 in helminth infections, and because basophils are capable of inducing Th2 differentiation of CD4(+) T cells and IgE isotype switching in B cells, we hypothesized that basophils function to amplify type 2 immune responses in chronic helminth infection. To test this, we evaluated basophil function using the Litomosoides sigmodontis filaria model of chronic helminth infection in BALB/c mice. Time-course studies showed that eosinophilia, parasite Ag-specific CD4(+) T cell production of IL-4 and IL-5 and basophil activation and IL-4 production in response to parasite Ag all peak late (6-8 wk) in the course of L. sigmodontis infection, after parasite-specific IgE has become detectable. Mixed-gender and single-sex worm implantation experiments demonstrated that the relatively late peak of these responses was not dependent on the appearance of circulating microfilariae, but may be due to initial low levels of parasite Ag load and/or habitation of the developing worms in the pleural space. Depletion of basophils throughout the course of L. sigmodontis infection caused significant decreases in total and parasite-specific IgE, eosinophilia, and parasite Ag-driven CD4(+) T cell proliferation and IL-4 production, but did not alter total worm numbers. These results demonstrate that basophils amplify type 2 immune responses, but do not serve a protective role, in chronic infection of mice with the filarial nematode L. sigmodontis.

Early recruitment of natural CD4+Foxp3+ Treg cells by infective larvae determines the outcome of filarial infection

Human helminth infections are synonymous with impaired immune responsiveness indicating suppression of host immunity. Using a permissive murine model of filariasis, Litomosoides sigmodontis infection of inbred mice, we demonstrate rapid recruitment and increased in vivo proliferation of CD4(+)Foxp3(+) Treg cells upon exposure to infective L3 larvae. Within 7 days post-infection this resulted in an increased percentage of CD4(+)T cells at the infection site expressing Foxp3. Antibody-mediated depletion of CD25(+) cells prior to infection to remove pre-existing 'natural' CD4(+)CD25(+)Foxp3(+) Treg cells, while not affecting initial larval establishment, significantly reduced the number of adult parasites recovered 60 days post-infection. Anti-CD25 pre-treatment also impaired the fecundity of the surviving female parasites, which had reduced numbers of healthy eggs and microfilaria within their uteri, translating to a reduced level of blood microfilaraemia. Enhanced parasite killing was associated with augmented in vitro production of antigen-specific IL-4, IL-5, IL-13 and IL-10. Thus, upon infection filarial larvae rapidly provoke a CD4(+)Foxp3(+) Treg-cell response, biasing the initial CD4(+) T-cell response towards a regulatory phenotype. These CD4(+)Foxp3(+) Treg cells are predominantly recruited from the 'natural' regulatory pool and act to inhibit protective immunity over the full course of infection.

Helminth Infection withLitomosoides sigmodontisInduces Regulatory T Cells and Inhibits Allergic Sensitization, Airway Inflammation, and Hyperreactivity in a Murine Asthma Model

Numerous epidemiological studies have shown an inverse correlation between helminth infections and the manifestation of atopic diseases, yet the immunological mechanisms governing this phenomenon are indistinct. We therefore investigated the effects of infection with the filarial parasite Litomosoides sigmodontis on allergen-induced immune reactions and airway disease in a murine model of asthma. Infection with L. sigmodontis suppressed all aspects of the asthmatic phenotype: Ag-specific Ig production, airway reactivity to inhaled methacholine, and pulmonary eosinophilia. Similarly, Ag-specific recall proliferation and overall Th2 cytokine (IL-4, IL-5, and IL-3) production were significantly reduced after L. sigmodontis infection. Analysis of splenic mononuclear cells and mediastinal lymph nodes revealed a significant increase in the numbers of T cells with a regulatory phenotype in infected and sensitized mice compared with sensitized controls. Additionally, surface and intracellular staining for TGF-beta on splenic CD4(+) T cells as well as Ag-specific TGF-beta secretion by splenic mononuclear cells was increased in infected and sensitized animals. Administration of Abs blocking TGF-beta or depleting regulatory T cells in infected animals before allergen sensitization and challenges reversed the suppressive effect with regard to airway hyperreactivity, but did not affect airway inflammation. Despite the dissociate results of the blocking experiments, these data point toward an induction of regulatory T cells and enhanced secretion of the immunomodulatory cytokine TGF-beta as one principle mechanism. In conclusion, our data support the epidemiological evidence and enhance the immunological understanding concerning the impact of helminth infections on atopic diseases thus providing new insights for the development of future studies.

CTLA-4 and CD4+CD25+ Regulatory T Cells Inhibit Protective Immunity to Filarial Parasites In Vivo

The T cell coinhibitory receptor CTLA-4 has been implicated in the down-regulation of T cell function that is a quintessential feature of chronic human filarial infections. In a laboratory model of filariasis, Litomosoides sigmodontis infection of susceptible BALB/c mice, we have previously shown that susceptibility is linked both to a CD4+ CD25+ regulatory T (Treg) cell response, and to the development of hyporesponsive CD4+ T cells at the infection site, the pleural cavity. We now provide evidence that L. sigmodontis infection drives the proliferation and activation of CD4+ Foxp3+ Treg cells in vivo, demonstrated by increased uptake of BrdU and increased expression of CTLA-4, Foxp3, GITR, and CD25 compared with naive controls. The greatest increases in CTLA-4 expression were, however, seen in the CD4+ Foxp3- effector T cell population which contained 78% of all CD4+ CTLA-4+ cells in the pleural cavity. Depletion of CD25+ cells from the pleural CD4+ T cell population did not increase their Ag-specific proliferative response in vitro, suggesting that their hyporesponsive phenotype is not directly mediated by CD4+ CD25+ Treg cells. Once infection had established, killing of adult parasites could be enhanced by neutralization of CTLA-4 in vivo, but only if performed in combination with the depletion of CD25+ Treg cells. This work suggests that during filarial infection CTLA-4 coinhibition and CD4+ CD25+ Treg cells form complementary components of immune regulation that inhibit protective immunity in vivo.

Ultrasonography in filaria-infected rodents: detection of adult Litomosoides sigmodontis and Brugia malayi filariae

To evaluate the usefulness of ultrasonography (USG) in the detection of adult filariae in rodents. Wuchereria bancrofti are frequently detected using USG in humans, whereas adult Brugia malayi have not been so far. A Meriones unguiculatus with Litomosoides sigmodontis infection was examined to visualize adult filariae of a similar length as W. bancrofti. Similarly, three Mastomys coucha, infected with B. malayi, were examined using USG to verify whether the adult worms, which are far smaller than W. bancrofti and L. sigmodontis, can be located using USG in the animals. Adult L. sigmodontis were detected using USG in the pleural cavity of M. unguiculatus, and in M. coucha adult B. malayi were visualized in the hearts, lungs, axillary lymph nodes and scrotum. Ultrasound findings were verified by dissection of the rodents. Although adult B. malayi are far smaller than L. sigmodontis and W. bancrofti, they can be detected using USG in rodents. USG may serve as an adjunctive tool to support parasitological examinations and can add information on filarial infections at any time point of an observation period, particularly in cryptic infections and without the need for invasive measures or killing of the rodent. Thus, USG can support the early detection of macrofilaricidal activities of new compounds and can be used to determine the location of adult worms in the animals. It is possible to give a rough estimate of the number of adult worms, but determination of the exact numbers of adult filariae in various locations is impossible with USG.

F4/80+ Alternatively Activated Macrophages Control CD4+ T Cell Hyporesponsiveness at Sites Peripheral to Filarial Infection

Both T cells and APC have been strongly implicated in the immune suppression observed during filarial nematode infections, but their relative roles are poorly understood, particularly in regard to timing and locality of action. Using Litomosoides sigmodontis infection of susceptible BALB/c mice, we have studied the progression of filarial immunosuppression leading to patent infection with blood microfilaremia. Patent infection is associated with decreased immune responsiveness in the draining thoracic lymph nodes (tLN) and intrinsically hyporesponsive CD4+ T cells at the infection site. We now show that we are able to separate, both in time and space, different suppressive mechanisms and cell populations that contribute to filarial hyporesponsiveness. L. sigmodontis infection recruited a F4/80+ population of alternatively activated macrophages that potently inhibited Ag-specific CD4+ T cell proliferative responses even in the presence of competent naive APC. T cell responsiveness was partially restored by neutralizing TGF-beta, but not by blocking IL-10 or CTLA-4 signaling. During prepatent infection, the macrophage population was restricted to the infection site. However, once infection became patent with systemic release of microfilariae, the suppressive macrophage activity extended peripherally into the tLN. In contrast, the hyporesponsive CD4+ T cell phenotype remained localized at the infection site, and the tLN CD4+ T cell population recovered full Ag responsiveness in the absence of suppressive macrophages. Filarial immunosuppression, therefore, evolves over time at sites increasingly distal to infection, and the mechanisms of filarial down-regulation are dependent on proximity to the infection site.

IL-4 is required to prevent filarial nematode development in resistant but not susceptible strains of mice

The murine Litomosoides sigmodontis model of filarial infection provides the opportunity to elucidate the immunological mechanisms that determine whether these nematode parasites can establish a successful infection or are rejected by the mammalian host. BALB/c mice are fully susceptible to L. sigmodontis infection and can develop patent infection, with the microfilarial stage circulating in the bloodstream. In contrast, mice on the C57BL background are largely resistant to the infection and never produce a patent infection. In this study, we used IL-4 deficient mice on the C57BL/6 background to address the role of IL-4 in the development of L. sigmodontis parasites in a resistant host. Two months after infection, adult worm recovery and the percentage of microfilaraemic mice in infected IL-4 deficient mice were comparable with those of the susceptible BALB/c mice while, as expected, healthy adults were not recovered from wild type C57BL/6 mice. The cytokine and antibody responses reveal that despite similar parasitology the two susceptible strains (BALB/c and IL-4 deficient C57BL/6) have markedly different immune responses: wild type BALB/c mice exhibit a strong Th2 immune response and the IL-4 deficient C57BL/6 mice exhibit a Th1 response. We also excluded a role for antibodies in resistance through infection of B-cell deficient C57BL/6 mice. Our data suggest that the mechanisms that determine parasite clearance in a resistant/non-permissive host are Th2 dependent but that in a susceptible/permissive host, the parasite can develop in the face of a Th2 dominated response.

Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility

Intracellular bacteria have been described in several species of filarial nematodes, but their relationships with, and effects on, their nematode hosts have not previously been elucidated. In this study, intracellular bacteria were observed in tissues of the rodent parasite Litomosoides sigmodontis by transmission electron microscopy and by immunohistochemistry using antiendobacterial heat shock protein-60 antisera. Molecular phylogenetic analysis of the bacterial 16S ribosomal RNA gene, isolated by PCR, showed a close relationship to the rickettsial Wolbachia endobacteria of arthropods and to other filarial intracellular bacteria. The impact of tetracycline therapy of infected rodents on L. sigmodontis development was analyzed in order to understand the role(s) these bacteria might play in filarial biology. Tetracycline therapy, when initiated with L. sigmodontis infection, eliminated the bacteria and resulted in filarial growth retardation and infertility. If initiated after microfilarial development, treatment reduced filarial fertility. Treatment with antibiotics not affecting rickettsial bacteria did not inhibit filarial development. Acanthocheilonema viteae filariae were shown to lack intracellular bacteria and to be insensitive to tetracycline. These results suggest a mutualistic interaction between the intracellular bacteria and the filarial nematode. Investigation of such a mutualism in endobacteria-containing human filariae is warranted for a potential chemotherapeutic exploitation.