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Interleukin-4 influences the production of microfilariae in a mouse model of Brugia infection
Abstract
Sub-cutaneous infection of interleukin (IL)-4-/- mice on the BALB/c background with third stage larva (L3) of Brugia pahangi revealed an altered cytokine profile consistent with the absence of the Th2 promoting cytokine IL-4. Splenocytes from IL-4-/- mice secreted significantly more antigen (Ag)-specific IL-2 and interferon-gamma and significantly less Ag-specific IL-5, compared to those from L3-infected wild-type mice. However, levels of Ag-specific IL-13 were similar between groups. Despite the alteration in immune responses, there was no significant difference in recovery of developing worms from the peritoneal cavity of the two strains of mice at any time postinfection. However, at later time points of infection, the IL-4-/- mice contained large numbers of microfilariae (Mf) in the peritoneal cavity while the wild-type mice contained comparatively few Mf. The differences in Mf levels appear to relate to differences in worm fecundity in the two strains of mice, with adult female worms from the wild-type mice containing few developing Mf. Moreover, implantation of sexually mature adult female worms into the peritoneal cavity of both strains of mice resulted in equal levels of Mf, confirming that the primary role of IL-4 is to limit fecundity during the maturation phase of infection.
... All mouse experiments were performed with male BALB/c mice, 6-8 weeks of age, and animals were maintained in filter top cages for the duration of the experiments. A transplant model was used to study the response elicited by B. pahangi adult worms, exactly as described previously [17,18]. In brief, adult female worms were removed from the peritoneal cavity of infected jirds, rinsed in HBSS and 10 worms transplanted into the peritoneal cavity of each of four anaesthetized BALB/c mice. ...
... To ensure that the infection was successful, the remaining six animals in this experiment were sacrificed at day 6 p.i. and the numbers of developing larvae were assessed by lavage of the peritoneal cavity with HBSS. Worm recoveries were variable 0-79% (mean 35±27%) in keeping with previous data from this model system [18], but five out of six animals contained developing worms. In addition, the peritoneal washings from infected animals showed evidence of a pronounced cellular infiltrate, consistent with the presence of worms. ...
... The BALB/c mouse is semi-permissive for B. pahangi in as much as infection with L3 gives rise to only a few adult worms and these do not produce microfilariae. However L3 and developing L4 can be recovered in reasonable numbers at early time points of infection [30], as can transplanted adult worms [18]. This model was selected for imaging over the more permissive jird for several reasons: 1) the availability of established protocols for imaging, efficacy and pharmacokinetic studies in mice, 2) the smaller size of mice compared to jirds increases the imaging capacity and reduces the compound requirement for dosing, 3) use of inbred mice compared to outbred jirds reduces variability, thus allowing smaller group sizes, and 4) the availability of antibodies to mouse proteins and genetically engineered mouse strains expands the downstream applications of the model. ...
Helminth parasites remain a major constraint upon human health and well-being in many parts of the world. Treatment of these infections relies upon a very small number of therapeutics, most of which were originally developed for use in animal health. A lack of high throughput screening systems, together with limitations of available animal models, has restricted the development of novel chemotherapeutics. This is particularly so for filarial nematodes, which are long-lived parasites with a complex cycle of development. In this paper, we describe attempts to visualise the immune response elicited by filarial parasites in infected mice using a non-invasive bioluminescence imaging reagent, luminol, our aim being to determine whether such a model could be developed to discriminate between live and dead worms for in vivo compound screening. We show that while imaging can detect the immune response elicited by early stages of infection with L3, it was unable to detect the presence of adult worms or, indeed, later stages of infection with L3, despite the presence of worms within the lymphatic system of infected animals. In the future, more specific reagents that detect secreted products of adult worms may be required for developing screens based upon live imaging of infected animals.
... Remarkably, for tissue helminths such as filarial nematodes that inhabit the skin, lymphatics or body cavities, the role of type 2 cytokines is still not resolved. Recent studies have suggested that, as with intestinal parasites, IL-4 and/or IL-13 may be important for the elimination of filarial parasites (Babu et al., 2000;Devaney et al., 2002;Volkmann et al., 2001). These studies are complicated by the fact that different cytokine pathways may be required to varying degrees at different stages of infection and that immune mechanisms may differ depending on the susceptibility/permissiveness of the murine host. ...
... C57BL/6 mice with L. sigmodontis. In contrast, Devaney et al. (2002) and Volkmann et al. (2001) using BALB/c mice found no effect of IL-4 on the development of infective larvae to adulthood using Brugia pahangi and L. sigmodontis, respectively. Interestingly, we also found no significant difference in adult recovery between WT and IL-4 deficient mice on the BALB/c background (unpublished observation). ...
... One consistent finding among all these studies is that once infection is established and the parasite has succeeded in achieving sexual maturity, IL-4 is the key determinant in the level of microfilariae. However, the data point to an impact on worm fecundity rather that to a direct effect on microfilaria survival (Devaney et al., 2002;Volkmann et al., 2001). ...
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.
... Adult worms were removed from the peritoneal cavity of infected jirds and rinsed in HBSS. 10 adult female worms were transplanted into the peritoneal cavity of each of ten male BALB/c mouse using standard methods [27,28]. Adult worms were transferred into the peritoneal cavity of anaesthetized mice using a blunted glass hook. ...
... In the first of these studies, pharmacokinetic analysis showed that the plasma levels of drug reached a maximum of 52,506 nM at 0.25 h post-dosing following a single dose of 50 mg/Kg delivered by the intra-peritoneal route. As a 24 h exposure to 250 nM NVP-AUY922 had a significant deleterious effect on adult female worms, we were encouraged to test this compound in vivo, using an adoptive transplant system in which viable adult worms are transplanted into the peritoneal cavity of mice [27,28]. In previous experiments with NVP-AUY922 administered in a similar dosing regimen to nude mice containing xeno-grafted tumors, weight loss was observed [38]. ...
Novel drugs are required for the elimination of infections caused by filarial worms, as most commonly used drugs largely target the microfilariae or first stage larvae of these infections. Previous studies, conducted in vitro, have shown that inhibition of Hsp90 kills adult Brugia pahangi. As numerous small molecule inhibitors of Hsp90 have been developed for use in cancer chemotherapy, we tested the activity of several novel Hsp90 inhibitors in a fluorescence polarization assay and against microfilariae and adult worms of Brugia in vitro. The results from all three assays correlated reasonably well and one particular compound, NVP-AUY922, was shown to be particularly active, inhibiting Mf output from female worms at concentrations as low as 5.0 nanomolar after 6 days exposure to drug. NVP-AUY922 was also active on adult worms after a short 24 h exposure to drug. Based on these in vitro data, NVP-AUY922 was tested in vivo in a mouse model and was shown to significantly reduce the recovery of both adult worms and microfilariae. These studies provide proof of principle that the repurposing of currently available Hsp90 inhibitors may have potential for the development of novel agents with macrofilaricidal properties.
... We have previously shown that absence of IL-4 does not effect the rate of Mf clearance in slow-clearing (C57BL/6 × 129) mice (15). This is corroborated by recent studies showing that development of B. pahangi L3 into Mf-producing adult worms and Mf levels are enhanced in IL-4 -/-BALB/c mice; however, the increase in Mf is a function of increased adult worm fecundity and not Mf survival (16). In contrast, Th2 responses correlated with Mf clearance of the bovine filarial nematode Setaria digitata in CBA /J mice (17). ...
... However, our data also raise the question of why Mf induce IFN-γ, particularly as presently there is little evidence that type 2 responses are lethal to Mf. A possible explanation is that Mf-induced IFN-γ production is an elaborate evasion mechanism used to down-regulate type 2 responses that can decrease adult worm fecundity and which are also potentially lethal to other parasite stages (16,21). Immune responses that are capable of protecting against each stage of B. malayi in humans are not yet defined. ...
Previous research has shown that Brugia malayi microfilariae (Mf) primarily induce type 1 cytokine production, and that in-vitro nitric oxide (NO) can mediate Mf killing. This study addresses the role of interferon (IFN)-gamma-mediated immune responses in the clearance of Mf from fast-clearing (CBA/Ca) and slow-clearing (C57Bl/6) mouse strains. Analysis of spleen cell cytokine production at early timepoints p.i. showed that Mf-induced IFN-gamma and nitrite (NO-) levels were significantly greater in CBA/Ca mice than C57Bl/6 mice. However, in-vivo neutralization of IFN-gamma or inhibition of NO- production in CBA/Ca mice did not alter Mf survival kinetics. Similarly, the rate of Mf clearance in both C57Bl/6 mice lacking the IFN-gamma gene and (C57Bl/6 x 129) mice deficient in the receptor for IFN-gamma was similar to that of wild-type animals. Furthermore, the dramatic abrogation of NO- production in IFN-gammaR-/- mice suggests that Mf clearance in slow-clearing mouse strains is also independent of NO- production. Thus, in both rapid-clearing and slow-clearing mouse strains, IFN-gamma-mediated mechanisms are not a requirement for Mf clearance from the bloodstream.
... In almost all experimental animal models of filariasis protective immunity is defined essentially as an antiparasite response that mediates decreased development of parasites when challenged with infective larvae or enhanced clearance of Mf from circulation. Notwithstanding these limitations studies on protective immunity in animal models have offered very crucial insights [6,11,12,18,19,21,34]. More importantly, animal models offer the facility of correlating an in vitro immunological parameter with protective immunity since they can be challenged with different developmental stages of parasites of parasites, namely L3, Mf, adult worms to monitor their development/survival in vivo. ...
... The immunological factors, if any, that may have played a role in elimination of Mf in these hosts are not currently clear. Recently in mouse models using genetically modified strains of mice IL-4 has been shown to play a role in inhibiting the fecundity of adult filariasis worms [6,46]. More interestingly, IL-10 has been demonstrated to assist in persistence of Mf in circulation [18]. ...
Human filariasis caused by lymphatic dwelling nematodes, affecting 120 million persons worldwide, is a major public health problem. Efforts towards development of vaccines for such large tissue-dwelling nematodes depends significantly on identification and demonstration of protective immunity in the exposed population. Immunological studies conducted in human filariasis so far are essentially attempts to establish a correlation of the immune response phenotypes with presence or absence of filarial infections/disease in the host, and the cause-effect relationship between the observed immune responses in the host and protective immunity continues to be conjectural. This short review attempts to clarify the functional definition of protective immunity, problems associated with identification of putatively immune subjects in endemic areas, role of antibodies reactive to surface of microfilariae and larvae stages of filarial parasites and importance of undertaking immunological investigations on a longitudinal basis in different cohorts of subjects presenting with one or the features of infection and/or disease for more accurate delineation of protective immunity in human filariasis.
... 20 Itraconazole achieves lower CSF levels than fluconazole but was more effective in an animal model of CNS histoplasmosis. 23,24 High-dose fluconazole is better tolerated than itraconazole but has a known risk of treatment failure. 25 Combination therapy of amphotericin B and an azole drug failed to show improved outcomes compared to amphotericin B alone; therefore, it is not recommended. ...
We report a case of a Puerto Rican male with advanced AIDS who presented with multiple falls and pancytopenia. Magnetic resonance imaging (MRI) of the brain, as initial workup, revealed 2 ring-enhancing brain lesions. Initial cerebrospinal fluid analysis revealed minimal cells, mildly elevated protein, and no organism seen on gram stain. Due to prohibitive thrombocytopenia, brain biopsy was deferred. He had neither clinical nor radiographic improvement despite empiric therapy for both toxoplasmosis and bacterial abscesses. Indicated by pancytopenia, bone marrow (BM) aspiration was performed. Culture of BM aspirate grew Histoplasma capsulatum. Urine histoplasma antigen was markedly elevated. He was treated with liposomal amphotericin B (LamB) for progressive disseminated histoplasmosis with probable central nervous system involvement. Cerebrospinal fluid histoplasma antigen obtained after 2 months of LamB was detected. After prolonged course of LamB, he took itraconazole. Brain MRI at 7-month follow-up revealed significant improvement from baseline study.
... The rejection of the parasite in mice is mediated by the adaptive immune system as nude (athymic) or scid mice tolerate the entire B. malayi lifecycle (193,195). However, although parasite rejection does take place in immunocompetent mice, a similar proliferative defect to that in jirds can be seen, indicating that some suppressive mechanisms, possibly including Treg induction, occur (48,177 (196,197) IL-4 -/-C57BL/6 Adults i.p. Loss of proliferative defect in peritoneal lavage cells ...
Many helminth parasites are able to survive for long periods in
immunocompetent hosts. It has been suggested that the successful establishment of
helminths is in part due to the induction of regulatory T cells (Tregs) which suppress
anti-parasite immune responses. A possible mechanism by which parasites induce
Tregs would be production of TGF-β, which can induce immunosuppressive Treg
cells and also directly suppress immune responses. TGF-β homologues have been
identified in parasitic nematodes including Brugia malayi, Schistosoma mansoni and
Ancylostoma caninum. The B. malayi TGF-β homologue, Bm-TGH-2, has been
demonstrated to signal through the mammalian TGF-β receptor, which indicates the
TGF-β homologues may have a function in the host. We hypothesised that Treg
induction through parasite-derived TGF-β could be a potent method by which
parasites could evade the host immune system.
To address this, molecular techniques were first used to identify and
characterise the transcription of novel TGF-β homologues from the intestinal
parasites Haemonchus contortus, Heligmosomoides polygyrus, Nippostrongylus
brasiliensis and Teladorsagia circumcincta, showing that TGF-β homologues are
present in a wide range of parasites. TGF-β activity was then shown in products from
both S. mansoni and H. polygyrus, and indeed the H. polygyrus products were found
to induce Tregs through the TGF-β pathway. Antiserum against bacterially expressed
H. polygyrus TGF-β homologue (Hp-TGH-2) was produced, and used to probe H.
polygyrus excretory/secretory products, showing that Hp-TGH-2 is secreted.
Attempts were also made to express recombinant Bm-TGH-2 (B. malayi TGF-β
homologue) in insect cells, however the purified protein proved not to be functional.
- v -
In vivo mouse models of B. malayi infection were tested to examine the
phenotype of T cell responses at the site of infection. An accumulation was found of
CD4+Foxp3+CD25+CTLA-4hiCD103+ T cells, which resemble activated natural
Tregs, and which were suppressive in vitro. This proportion of Tregs at the site of
infection diminishes over time, however CD103 expression (which is associated with
activated Tregs) is increased on Tregs present at all timepoints up to day 21 postinfection,
indicating that although a growing effector response may outgrow Tregs
over time, the Treg population remains activated. Using an adoptive transfer model,
it was shown that the Treg induction could spread to other bystander responses. In
IL-4R-deficient mice, Treg accumulation was unaffected, indicating alternatively
activated macrophages (which are absent in these mice) are not required for Treg
induction.
... The timing of these events is indicative of Tindependent responses but there is also good evidence that IL4 or IL13 driven pathways are necessary for the control of microfilaraemia following natural challenge with L. sigmodontis L3 in both susceptible and resistant strains (59,61,75,76). Some argue that the IL4 responses negatively impact on fecundity rather than being responsible for direct attack on microfilariae (59,61,77). However, IL4 may also act through promoting antibody dependent cellular cytotoxicity responses that can mediate clearance of microfilariae (78). ...
Filarial infections remain a major public health and socio-economic problem across the tropics, despite considerable effort to reduce disease burden or regionally eliminate the infection with mass drug administration programmes. The sustainability of these programmes is now open to question owing to a range of issues, not least of which is emerging evidence for drug resistance. Vaccination, if developed appropriately, remains the most cost-effective means of long-term disease control. The rationale for the feasibility of vaccination against filarial parasites including onchocerciasis (river blindness, Onchocerca volvulus) and lymphatic filariasis (Wuchereria bancrofti or Brugia malayi) is founded on evidence from both humans and animal models for the development of protective immunity. Nonetheless, enormous challenges need to be faced in terms of overcoming parasite-induced suppression without inducing pathology as well as the need to both recognize and tackle evolutionary and ecological obstacles to successful vaccine development. Nonetheless, new technological advances in addition to systems biology approaches offer hope that optimal immune responses can be induced that will prevent infection, disease and/or transmission.
... For example, in Schistosoma haematobium infections, egg output declines in a T-cell-dependent manner [61], while fecundity of H. polygyrus is raised following anti-CD4 depletion [62]. IL-4 is an important component in the regulation of H. polygyrus egg production [63] and is also required to control microfilarial output in Brugia pahangi [64] and L. sigmodontis [65]. Anti-fecundity effects have been replicated by passive transfer of immune serum [66] and are also observed in a number of recombinant anti-helminth vaccination trials [67,68]. ...
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.
... This is consistent with models of eosinophil recruitment in the lung in which reduced but not absent eosinophilia is seen in both IL-4 deficient and IL-13 deficient mice, but double deficient mice completely fail to recruit eosinophils [31]. Devaney et al. [32] also recently found significantly reduced eosinophils in IL-4 –/– BALB/c mice infected with B. pahangi. However , these results differed somewhat from our study as they found significantly reduced total cell recruitment in the absence of IL-4 on the BALB/c background. ...
Mice exposed intraperitoneally to either adult or first larval stage (microfilaria) of the human nematode parasite Brugia malayi display polarized cytokine responses. We have used this model to investigate the impact of altered cytokine profiles on inflammatory cell recruitment patterns in vivo. Here we demonstrate that Th2-inducing adult parasites drive the recruitment of eosinophils and macrophages after implant into the murine peritoneal cavity whereas Th1-inducing microfilaria do not. The underlying mechanism of recruitment was further defined by use of mice deficient in the key Th2 cytokines IL-4 or IL5 and mice that lack T cells (nude mice). Recruitment dynamics differed in IL-4 and IL-5 deficient mice, showing reduced or absent eosinophilia. These data emphasize the pivotal role of these cytokines in shaping the cellular profile of inflammatory responses. Surprisingly, the absence of T cells failed to influence inflammatory cell recruitment indicating that recruitment signals are provided by other cell types.
... What is clear, however, is that Mf in mice is controlled by IL-4 and IL-13: depletion or knockout of these cytokines [66,67] or their common receptor (IL-4R Ϫ/Ϫ BALB/c mice; data not shown) increases microfilariae levels у10-fold, compared with levels in wild-type. These results are paralleled by evidence that Brugia malayi microfilariae injected into mice are cleared by type 2 [68,69], not type 1 [70], effector mechanisms. Of further interest, injected Mf induce IFN-g production on their own [71,72]. ...
Coinfections are common in natural populations, and the literature suggests that helminth coinfection readily affects how
the immune system manages malaria. For example, type 1-dependent control of malaria parasitemia might be impaired by the type
2 milieu of preexisting helminth infection. Alternatively, immunomodulatory effects of helminths might affect the likelihood
of malarial immunopathology. Using rodent models of lymphatic filariasis (Litomosoides sigmodontis) and noncerebral malaria (clone AS Plasmodium chabaudi chabaudi), we quantified disease severity, parasitemia, and polyclonal splenic immune responses in BALB/c mice. We found that coinfected
mice, particularly those that did not have microfilaremia (Mf−), had more severe anemia and loss of body mass than did mice with malaria a;lone. Even when controlling for parasitemia,
malaria was most severe in Mf− coinfected mice, and this was associated with increased interferon-γ responsiveness. Thus, in Mf− mice, filariasis upset a delicate immunological balance in malaria infection and exacerbated malariainduced immunopathology.
... What is clear, however, is that Mf in mice is controlled by IL-4 and IL-13: depletion or knockout of these cytokines [66, 67] or their common receptor (IL-4R / BALB/c mice; data not shown) increases microfilariae levels 10-fold, compared with levels in wild-type. These results are paralleled by evidence that Brugia malayi microfilariae injected into mice are cleared by type 2 [68, 69], not type 1 [70], effector mechanisms. Of further interest, injected Mf induce IFN-g production on their own [71, 72]. ...
The impact of demographic characteristics, phase of the menstrual cycle, use of hormonal contraceptives, and concomitant lower
genital-tract infections on cervicovaginal inflammatory cells was assessed in 967 women, 654 of whom were infected with human
immunodeficiency virus type 1 (HIV-1). Cervicovaginal lavage (CVL) fluid was evaluated for total white blood cell (WBC), polymorphonuclear
leukocyte, and monocyte counts. HIV-1 infection was not associated with statistically significant differences in numbers of
inflammatory cells in CVL fluid except in 1 group—HIV-1-infected women with Chlamydia trachomatis infection had a 0.43 log10 higher WBC count than their HIV-uninfected, chlamydia-positive counterparts (P = .04). Younger age and use of progesterone-based hormonal contraceptives were independently associated with increased numbers
of inflammatory cells in CVL fluid. A 0.15–0.2 log10 increase in inflammatory cells was seen in black versus white and Hispanic women after adjustment for known potential confounders.
Progesterone-based contraceptives, younger age, and race have an independent effect on cervicovaginal inflammatory cells.
... What is clear, however, is that Mf in mice is controlled by IL-4 and IL-13: depletion or knockout of these cytokines [66, 67] or their common receptor (IL-4R / BALB/c mice; data not shown) increases microfilariae levels 10-fold, compared with levels in wild-type. These results are paralleled by evidence that Brugia malayi microfilariae injected into mice are cleared by type 2 [68, 69], not type 1 [70], effector mechanisms. Of further interest, injected Mf induce IFN-g production on their own [71, 72]. ...
Immunological and virological consequences of low-level viremia in human immunodeficiency virus (HIV) type 1-infected patients receiving highly active antiretroviral therapy (HAART) remain to be determined.
For 24 months, 101 HAART-treated, HIV-1-infected patients with HIV RNA levels </=200 copies/mL were followed prospectively: HIV RNA level and CD4 and CD8 cell counts were investigated every 3 months, and proviral DNA and T cell subsets were investigated every 6 months.
During follow-up, 33 patients had HIV RNA levels </=20 copies/mL at all visits (uVL patients), whereas 68 patients had HIV RNA levels >20 copies/mL at >/=1 visit (dVL patients) (median increase, 81 copies/mL [interquartile range, 37-480 copies/mL]). dVL patients had higher concentrations of CD8 cells, activated and memory T cells, and proviral DNA, compared with uVL patients (P<.05). A higher HIV RNA level was independently associated with reduced CD4 gain (P<.001). A higher HIV RNA level also was associated with increases in activated CD8(+)CD38(+) and CD8(+)HLA-DR(+) cells (P<.05), and a higher level of activated CD8(+)CD38(+) cells was independently associated with reduced CD4 gain (P<.05). A higher proviral DNA level was associated with increases in CD4(+)CD45RA(-)CD28(-) effector cells and reductions in naive CD4(+)CD45RA(+)CD62L(+) and CD8(+)CD45RA(+)CD62L(+) cells (P<.05). Higher levels of activated CD4(+)HLA-DR(+) and early differentiated CD4(+)CD45RA(-)CD28(+) cells predicted increased risk of subsequent detectable viremia in patients with undetectable HIV RNA (P<.05).
These findings indicate that low-level viremia and proviral DNA are intimately associated with the immunological and virological equilibrium in patients receiving HAART.
... The large size of intrauterine stages (about 50 µM for egg stages and about 160 µM for Mf) do not limit the applicability of this novel assay since no special modification in the flowcytometer is required, the dimensions of standard flowcell provided by the manufacturers in FACSCalibur model was sufficient to perform the assays. We envisage a wide application for this new flowcytometry-based method of monitoring embryogenesis as well as antibody binding to intra-uterine stages in filariasis research: a) removal of endosymbionts in human hosts by doxycycline requires Antibody binding in chronic filariasis cases; administration of the drug over a period of several weeks (5) and there is a need for developing newer anti-rickettsial drugs for blocking embryogenesis/fecundity in adult filarial worms; the new method described in this communication can be effectively used for screening a large number of potential compounds/drugs; b) although doxycycline and tetracycline have been demonstrated to eliminate endosymbionts in adult filarial worms, the precise intrauterine stage at which embryogenesis is blocked is not known and can now be studied with this new assay; c) cytokines such as IL-4 and molecules of innate immunity such as TLR-4 have been demonstrated to play a role in regulating production of microfilaraemia in experimental hosts [4,10]. Adult filarial worms can now be implanted in mice made deficient for specific cytokine gene expression or transgenic for such host molecules to monitor their role in embryogenesis; d) several filarial antigens have been cloned, sequenced and expressed in recent years and some have been used as putative vaccine candidates in experimental models [11]; e) high reactivity of antibodies in human filariasis sera to a select population of Mf (Fig 5a &5b) could be due to polymorphic antigens expressed on Mf sheath described by us several years ago (12). ...
In the absence of intermediate animal hosts, the process of embryogenesis leading to fecundity of adult female filarial worms is very critical for persistence of these obligate parasites in human communities. Embryogenesis in adult female filarial parasites involves fertilization of eggs or oocytes by sperms and their subsequent development into motile microfilariae inside the uterine cavity of worms. Development of assays for monitoring embryogenesis in adult female worms is a critical requirement in filariasis research--filarial worms are known to harbour endosymbionts such as Wolbachia which play a significant role in fecundity. Tetracycline or doxycycline treatment of the infected hosts effectively eliminates the endosymbionts resulting in inhibition of embryogenesis in female worms. Currently, inhibition of embryogenesis in adult filarial worms can be monitored only by microscopic examination of in vitro harvested intrauterine stages.
Adult female filarial worms of bovine filarial parasites, Setaria digitata were collected from the peritoneum of infected animals and intrauterine stages were harvested in culture medium and were analyzed for forward and side scatter by flowcytometry using a BD FACS Calibur. Different populations were gated, sorted and identified by phase microscopy. Binding of biotinylated lectins to intra uterine stages was monitored using FITC labeled Avidin and monitored by flow cytometry of gated populations. Similarly, binding of antibodies in human filarial sera to intrauterine stages was monitored using FITC labeled anti-human immunoglobulins.
The forward and side scatter for intrauterine stages delineated 3 distinct populations labeled as R1, R2 and R3. The three populations were sorted and identified to be a) fully stretched microfilariae, b) early and c) late developmental stages of eggs respectively. Lectins such as Wheat Germ agglutinin or Concanavalin-A were found to bind strongly to egg stages and less prominently to intra-uterine microfilariae. Similarly the binding of antibodies in filarial sera to the three intra-uterine stages could also be precisely quantified.
The manuscript reports a novel flow cytometry based method to monitor progression of embryogenesis in adult filarial worms. Apart from relative quantification of different intra uterine developmental stages, the assay allows quantitative binding of lectins and antibodies to each of the intrauterine stages. It may now be possible to quantify levels of antibodies in infected and immune hosts to monitor anti-fecundity immunity in filariasis--the assay can thus be used as a powerful tool for drug development and in immunological studies in human and experimental filariasis.
... Further, BALB/c mice deficient in IL-4, IL-5, or IL-4Ra (unable to respond to IL-4 or IL-13) present with levels of microfilariae 100 times higher than wild-type controls [31,32]. This evidence that type 2 cytokines can control microfilarial levels is consistent with studies on Brugia species [33]. Although the data began to build a convincing argument for Th2 control of filarial infections, the picture that emerged proved more complex. ...
River blindness is a seriously debilitating disease caused by the filarial parasite Onchocerca volvulus, which infects millions in Africa as well as in South and Central America. Research has been hampered by a lack of good animal models, as the parasite can only develop fully in humans and some primates. This review highlights the development of two animal model systems that have allowed significant advances in recent years and hold promise for the future. Experimental findings with Litomosoides sigmodontis in mice and Onchocerca ochengi in cattle are placed in the context of how these models can advance our ability to control the human disease.
Throughout the lifespan of an individual, the immune system undergoes complex changes while facing novel and chronic infections. Helminths, which infect over one billion people and impose heavy livestock productivity losses, typically cause chronic infections by avoiding and suppressing host immunity. Yet, how age affects immune responses to lifelong parasitic infection is poorly understood. To disentangle the processes involved, we employed supervised statistical learning techniques to identify which factors among haematopoietic stem and progenitor cells (HSPC), and both innate and adaptive responses regulate parasite burdens and how they are affected by host age. Older mice harboured greater numbers of the parasites' offspring than younger mice. Protective immune responses that did not vary with age were dominated by HSPC, while ageing specifically eroded adaptive immunity, with reduced numbers of naïve T cells, poor T cell responsiveness to parasites, and impaired antibody production. We identified immune factors consistent with previously-reported immune responses to helminths, and also revealed novel interactions between helminths and HSPC maturation. Our approach thus allowed disentangling the concurrent effects of ageing and infection across the full maturation cycle of the immune response and highlights the potential of such approaches to improve understanding of the immune system within the whole organism.
SUMMARY Nematodes are amongst the most successful and abundant organisms on the planet with approximately 30 000 species described, although the actual number of species is estimated to be one million or more. Despite sharing a relatively simple and invariant body plan, there is considerable diversity within the phylum. Nematodes have evolved to colonize most ecological niches, and can be free-living or can parasitize plants or animals to the detriment of the host organism. In this review we consider the role of heat shock protein 90 (Hsp90) in the nematode life cycle. We describe studies on Hsp90 in the free-living nematode Caenorhabditis elegans and comparative work on the parasitic species Brugia pahangi, and consider whether a dependence upon Hsp90 can be exploited for the control of parasitic species.
Mouse models of Brugia infection have provided much useful quantitative and qualitative information on the immune response elicited by different life cycle stages of filarial worms. Many parallels exist between the immune response in the mouse and the infected human and in this review we highlight areas of topical interest, including the induction of specific cytokine resposes and their role in immunomodulation and protective immunity. These studies have reinforced the concept that different life cycle stages of filarial parasites each have their own mechanism of modulating responses so that potentially inflammatory IFN-γ responses are downregulated. While the precise mechanisms of protective immunity remain to be defined, studies in the mouse have suggested novel pathways, including a possible role for granulocytes.
Die vorliegende Arbeit befasst sich mit der Synthese und den Wirkungen eines exkretorisch-sekretorischen Produkts, Juv-p120, aus der Nagetierfilarie Litomosoides sigmodontis, für das eine immunmodulatorische Funktion vermutet wurde.
Es handelt sich um ein hochgradig mit über Phosphatbrücken gebundenem Dimethylaminoethanol (DMAE) modifiziertes Molekül mit einer molekularen Masse von ca. 130 kDa. Es wurde von in vitro in proteinfreiem Milieu gehaltenen, 39 Tage alten präadulten Parasiten gewonnen. Es lässt sich mittels eines polyklonalen, in erster Linie gegen die Modifikation gerichteten Antiserums in Form einer Doppelbande im Bereich von 130 - 150 kDa nachweisen. Das Molekül wurde angereichert durch Filtration durch eine Membran mit einer Ausschlussgrenze von 100 kDa.
Die nachfolgenden Untersuchungen wurden teils vergleichend mit Mastomys coucha und BAL/c-Mäusen durchgeführt. In M. coucha betrug die Rückfindungsrate 120 Tage nach quantitativer L. sigmodontis-Infektion 23 %, bei BALB/c-Mäusen 80 Tage p. i. 5 %. Daneben fanden sich bei Mäusen in etwa gleicher Menge tote, abgekapselte Parasiten. M. coucha entwickelten eine ausgeprägte Parasitämie, in den Mäusen ließen sich nur vereinzelt Mikrofilarien nachweisen.
Juv-p120 wird von L3, L4 sowie in geringen Mengen von jungen Weibchen synthetisiert und konnte im Blutplasma L. sigmodontis-infizierter BALB/c-Mäuse 31 bis 51 Tage p. i. nachgewiesen werden. Antikörper gegen Juv-p120 ließen sich in infizierten Mäusen ab Tag 14 p. i., in M. coucha ab Tag 35 p.i., in geringen Mengen noch 70 und 160 Tage p. i. nachweisen. Mit angereichertem Juv-p120 (nÜP) immunisierte M. coucha zeigten nach Belastungsinfektion eine tendenziell reduzierte Parasitämie. Die Wurmzahl war nicht beeinträchtigt, doch entwickelten die weiblichen Würmer einen erhöhten Anteil an pathologisch veränderten Embryonen. Bei immunisierten Mäusen ließ sich kein Einfluss auf eine Belastungsinfektion nachweisen.
Milzlymphozyten infizierter M. coucha proliferierten anders als die nicht infizierter Tiere nach Stimulation mit ConA in der Präpatenz kaum, in der Patenz und Postpatenz nicht mehr. nÜP induzierte lediglich in der frühen Patenz eine Proliferation. Zellen aus infizierten BALB/c-Mäusen reagierten auf ConA in der frühen Präpatenz und beginnenden Patenz schwächer als nicht infizierte Kontrollen, proliferierten aber dann mit der Infektionsdauer zunehmend nach Stimulation mit nÜP.
Die Transkription von Genen, kodierend für IFNγ, IL-2, IL-4, IL-5, IL-10 und IL-13, wurde in Milzlymphozyten L. sigmodontis-infizierter BALB/c-Mäuse nach Stimulation mit ConA und nÜP mittels einer semiquantitativen RT-PCR bis 160 Tage p. i. bestimmt. Die Stimulierung mit ConA induzierte in allen Fällen eine gesteigerte Gentranskription, wobei IFNγ-, IL-10- und IL-13-Gentranskripte in der frühen Präpatenz und in der Postpatenz gesteigert waren. IL-2- und IL-4-Gentranskripte waren bei Zellen aus der frühen Präpatenz im Vergleich zu anderen Infektionsphasen eher erniedrigt. Im Fall des IL-5-Gens ließ sich nur in der späten Präpatenz eine erhöhte Transkription nachweisen. nÜP steigerte die Transkription des IFNγ-Gens allenfalls in der Postpatenz, die des IL-2- und IL-4-Gens in der späten bzw. frühen Präpatenz, hatte bei den übrigen untersuchten Genen dagegen kaum einen Einfluss.
Um den Einfluss von Juv-p120 und P-DMAE auf die Proliferationsfähigkeit von Milzlymphozyten zu überprüfen, wurden Zellen infizierter M. coucha und BALB/c-Mäuse zu verschiedenen Zeitpunkten p. i. mit ConA im Beisein von nÜP (a), einem synthetischen, Juv-p120-unabhängigen, P-DMAE-modifizierten Peptid (b) sowie (c) dem nicht modifizierten Kontrollpeptid stimuliert. Das letztere Peptid hatte keinen Einfluss auf die Proliferation. Bei M. coucha-Milzlymphozyten ließ sich die Reaktion mit den verwendeten Mengen auch durch nÜP nicht signifikant modifizieren. Das P-DMAE-Peptid steigerte die Reaktion auf ConA in dosisabhängiger Weise bei Zellen aus der Postpatenz, die ansonsten refraktär gegen ConA waren. Maus-Milzlymphozyten wurden dagegen durch das P-DMAE-Peptid nicht in ihrer Proliferation auf ConA-Stimulierung beeinflusst, während nÜP die Reaktion von Zellen aus der Präpatenz supprimierte. Bei Lymphozyten aus der Patenz und Postpatenz führte es zu einer gesteigerten Proliferation. The thesis deals with the synthesis and effects of Juv-p120, a secretory-excretory product of the rodent filaria Litomosoides sigmodontis, which was supposed to act in an immunomodulatory manner. Juv-p120 is a protein of a molecular mass of approximately 130 kDa which is intensely postranslationally modified by dimethyl-aminoethanol (DMAE) bound by phosphate bonds. It was collected from 39 days old, preadult female worms, incubated in protein-free medium. Using a polyclonal rabbit antiserum which was mainly directed against the modification Juv-p120 could be demonstrated by immunoblotting as a double band of 130 - 150 kDa. The molecule was enriched by filtration of the incubation supernatant through a membrane with an exclusion limit of 100 kDa.
Subsequent studies were in part performed comparatively in Mastomys coucha and BALB/c mice as experimental hosts. The developmental rate in M. coucha, determined 120 days p. i., was 23 %, in BALB/c mice, determined 80 days p. i., 5 %. In addition mice contained almost the same numbers of dead, encapsulated parasites. M. coucha showed an intense parasitaemia whereas microfilariae in the mice were found only occasionally.
Juv-p120 was synthetized by L3 and L4 and, in small amounts, by young female worms and was demonstrated in the blood of L. sigmodontis-infected BALB/c mice 31 - 51 days p. i. Antibodies to Juv-p120 were found in infected mice as early as 14 days p. i. and occurred in M coucha at day 35 p.i. and in low concentration still 70 and 160 days p. i.. M. coucha and BALB/c-mice were immunized with enriched Juv-p120 (nÜP) and challenged with L3 of L. sigmodontis. Immunized M. coucha, as a tendency, showed reduced parasitaemia levels but numbers of adult parasites, found 120 days p. i., did not differ from controls. However, female parasites contained increased proportions of pathologically altered intrauterine stages. Immunized BALB/c mice did not differ from controls by parasitological data.
Spleen lymphocytes of L. sigmodontis infected M. coucha responded to ConA in contrast to uninfected only weakly by prolifertation and were found non-reactive during patency and postpatency. nÜP stimulation induced lymphocyte proliferation only in early patency. Cells of infected mice isolated during prepatency and early patency responded weaker to ConA than non infected controls but later on proliferated increasingly with time after infection.
Transcriptions of IFNγ, IL-2, IL-4, IL-5, IL-10 and IL-13 genes induced by ConA and nÜP stimulation were studied in infected BALB/c mice up to 160 days p. i. employing a semiquantitative RT-PCR. ConA induced enhanced transcription of the genes tested, whereby increased transcript levels of IFNγ, IL-10 and IL-13 genes were found during early patency and in the stage of postpatency. In case of IL-2 and IL-4 gene transcription was rather decreased during prepatency when compared to other phases of the infection. IL-5 gene transcripts were particularly enhanced in the late prepatency. nÜP caused increased IFNγ gene transcription only in cells isolated during postpatency. IL-2 and IL-4 gene transcripts were enhanced by nÜP during late and early prepatency, respectively. In case of the other genes tested, nÜP was ineffective.
To study the influence of Juv-p120 and DMAE on the proliferative capacity of spleen lymphocytes cells were isolated from L. sigmodontis infected M. coucha and BALB/c mice at various dates p.i. and exposed to ConA in the presence of (i) nÜP, (ii) a Juv-p120-independent synthetic P-DMAE-modified peptide and (iii) the non-modified control peptide. The latter peptide did not affect ConA-induced proliferation at all. Also nÜP in the applied concentrations failed to influence the response of M. coucha lymphocytes. However, P-DMAE-peptide increased the proliferative response of spleen cells isolated from postpatent M. coucha which were otherwise non-responsive to ConA in a dose dependent manner. In contrast, the proliferation of mouse lymphocytes was not influenced by P-DMAE-peptide, whereas nÜP depressed the response of mouse lymphocytes, isolated during prepatency, but enhanced the proliferation of cells of patent and postpatent mice which otherwise did not respond to ConA at all.
Cytokine (interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-5, IL-10) gene transcription in response to filarial antigens was determined in peripheral blood mononuclear cells of Brugia
malayi-infected Mastomys coucha in the course of untreated and chemotherapeutically abbreviated infections. Transcript levels in infected untreated animals suggest particular time courses for the various cytokines with ongoing parasite development and differing efficacies of female, male, microfilarial, and L3 antigens in inducing cytokine gene transcription. Gene transcription of both of Th1- and Th2-associated cytokines were initiated in the course of the infection in a manner that does not fit in a simple Th1–Th2 paradigm. IFN-γ and IL-4 gene transcripts prevailed during prepatency. In case of the other cytokine genes considered in the study, transcription in general peaked around beginning of patency. During the phase of increasing microfilaremia (approximately 120–180 days p. i.) cytokine gene transcription was generally decreased. Later on, when the parasitemia had leveled off, except IFN-γ, transcript levels often tended to increase. In chemotherapeutically treated animals, the outcome varied with the different efficacies of the drugs employed. The highly microfilaricidal cyclodepsipeptide BAY 44-4400 eliminated circulating microfilariae and partially sterilized adult worms without killing them. This kind of treatment hardly affected cytokine responses. In contrast, the therapy with Flubendazole®, a selectively macrofilaricidal benzimidazole, and particularly the application of CGP 20376, a highly efficient microfilaricidal and macrofilaricidal benzthiazole, resulted in enhanced transcription of the Th1-associated IFN-γ and IL-2 genes as well as of the Th2-associated IL-5 gene 2–3 months after treatment. IL-10 gene transcription seemed transiently increased after 1 month. There was no effect of any treatment on the IL-4 gene transcription.
Primary and secondary murine and human infections with Brugia malayi are characterized by substantial increases in levels of immunoglobulin E (IgE). To investigate whether this is necessary
for worm clearance, IgE−/− mice were subjected to primary- and secondary-infection protocols. Following a primary infection, IgE−/− mice displayed a profound deficit in their ability to clear an intraperitoneal injection of L3 infective-stage larvae in
comparison to wild-type counterparts and maintained substantial worm burdens as late as 10 weeks postinfection. Although viable
adult parasites were recovered at this late time point from IgE−/− mice, the majority of the mice remained free of microfilariae. IgE−/− cohorts subjected to a secondary-infection protocol were able to clear the challenge inoculation in an accelerated manner,
with kinetics similar to that observed in the wild-type animals. Analysis of the humoral response in IgE−/− mice following infection demonstrates a defect in IgG1 and IgG2a production, in addition to the expected lack of IgE. The
IgG1 deficiency is no longer evident following a secondary infection. These data imply that deficiencies other than IgE production
(i.e., IgG1 production) deficiency may be responsible for the increased permissiveness of IgE−/− mice as hosts following infection with B. malayi.
Approximately 30 years ago, researchers reported intracellular bacteria in filarial nematodes. These bacteria are relatives
of the arthropod symbiont Wolbachia and occur in many filarial nematodes, including Brugia pahangi and Brugia malayi. Wolbachia bacteria have been implicated in a variety of roles, including filaria development and fecundity and the pathogenesis of
lymphatic lesions associated with filarial infections. However, the role of the bacteria in worm biology or filarial disease
is still not clear. The present experiments support previous data showing that tetracycline eliminates or reduces Wolbachia bacteria in B. pahangi in vivo. The elimination of Wolbachia was closely linked to a reduction in female fecundity and the viability of both sexes, suggesting that the killing of Wolbachia is detrimental to B. pahangi. The gerbils treated with tetracycline showed reduced levels of interleukin-4 (IL-4) and IL-5 mRNA in renal lymph nodes and
spleens compared with the levels in B. pahangi-infected gerbils not treated with tetracycline. However, similar findings were noted in B. pahangi-infected gerbils treated with ivermectin, suggesting that the loss of circulating microfilariae, not the reduction of Wolbachia bacteria, was associated with the altered cytokine profile. Despite the change in T-cell cytokines, there was no difference
in the sizes of renal lymph nodes isolated from gerbils in each treatment group. Furthermore, the numbers, sizes, or cellular
compositions of granulomas examined in the lymphatics or renal lymph nodes did not differ with treatment. These data suggest
that Wolbachia may not play a primary role in the formation of lymphatic lesions in gerbils chronically infected with B. pahangi.
In order to understand natural resistance to filariasis, we compared Litomosoides sigmodontis primary infection of C57BL/6 mice, which eliminate the worms before patency, and BALB/c mice, in which worms complete their development and produce microfilariae. Our analysis over the first month of infection monitoredmigration of the infective larvae from the lymph nodes to the pleural cavity, where the worms settle. Although immune responses from the mouse strains differed from the outset, the duration of lymphatic migration (4 days) and filarial recovery rates were similar, thus confirming that the proportion of larvae that develop in the host species upon infection is not influenced by host genetic variability. The majority of worms reached the adult stage in both mouse strains; however, worm growth and molting were retarded in resistant C57BL/6 mice. Surprisingly, the only immune responses detected at 60 h postinfection occurred in the susceptible mice and only upon stimulation of cells from lymph nodes draining the inoculation site with infective larva extract: massive production of interleukin-6 (IL-6) and IL-5 (the latter cytokine was previously suspected to have an effect on L. sigmodontis growth). However, between days 10 and 30 postinfection, extraordinarily high levels of type 1 and type 2 cytokines and expansion of pleural leukocyte infiltration were seen in the resistant C57BL/6 mice, explaining the destruction of worms later. Our results suggest that events early in the infection determine susceptibility or resistance to subsequent microfilarial production and a parasite strategy to use specific immune responses to its own benefit.
Immune regulation by parasites is a global concept that includes suppression, diversion, and conversion of the host immune response to the benefit of the pathogen. While many microparasites escape immune attack by antigenic variation or sequestration in specialized niches, helminths appear to thrive in exposed extracellular locations, such as the lymphatics, bloodstream, or gastrointestinal tract. We review here the multiple layers of immunoregulation that have now been discovered in helminth infection and discuss both the cellular and the molecular interactions involved. Key events among the host cell population are dominance of the T-helper 2 cell (Th2) phenotype and the selective loss of effector activity, against a background of regulatory T cells, alternatively activated macrophages, and Th2-inducing dendritic cells. Increasingly, there is evidence of important effects on other innate cell types, particularly mast cells and eosinophils. The sum effect of these changes to host reactivity is to create an anti-inflammatory environment, which is most favorable to parasite survival. We hypothesize therefore that parasites have evolved specific molecular strategies to induce this conducive landscape, and we review the foremost candidate immunomodulators released by helminths, including cytokine homologs, protease inhibitors, and an intriguing set of novel products implicated in immune suppression.
Litomosoides sigmodontis is the only filaria which develops from infective larvae into microfilaria-producing adults in immunocompetent laboratory
mice. In this study we report that interleukin-4 knockout (IL-4 KO) mice have an up to 100-fold-higher and a significantly
prolonged microfilaremia compared to wild-type BALB/c mice, as well as 20 times more microfilariae in the thoracic cavity,
the site of infection. While worm development and adult worm persistence were equivalent in IL-4 KO and wild-type mice, the
fertility and length of adult female worms in IL-4 KO mice was clearly enhanced. The high susceptibility to microfilariae
in IL-4 KO mice required the presence of adult worms in a full infection cycle since microfilariae loads did not differ much
between IL-4 KO and wild-type mice when purified microfilariae were injected into mice. In addition, we found that eosinophilia
was diminished and immunoglobulin E (IgE) was absent in IL-4 KO mice. IgE, however, does not seem to be the essential factor
for microfilarial containment since microfilaremia was not elevated in B-cell KO mice. In conclusion, IL-4 is shown for the
first time to be essential for the control of microfilarial loads but not of adult worm loads in a fully permissive murine
filarial infection. IL-4 dependent effector pathways seem to operate on adult worms rather than directly on microfilariae.
Immunity to Onchocerca volvulus (Ov) infection is suggested by the presence of putatively immune (PI) subjects in a region of Ecuador in which Ov is endemic.
PI subjects were identified by traditional diagnostic methods combined with a polymerase chain reaction-based assay for Ov
DNA in skin snips. Responses of peripheral blood mononuclear cells (PBMC) from the PI group (n = 16) were compared with those of persons with active infection (microfiladermic [MF] subjects; n = 51). PBMC of PI subjects proliferated significantly more to Ov antigen (OvAg; P < .009) than did PBMC ofMF persons but less to streptolysin-O (P < .001). Cytokine analysis of PBMC culture supernatants revealed that PI subjects (n = 11) produced significantly more interferon-ã to OvAg than did those in the MF group (n = 18; P = .018), less interleukin (IL)-5 to nonparasite antigen (P = .003) and mitogen (P = .012), and less IL-I0 spontaneously (P = .016). Thus, immunity to Ov may in part be mediated by an antigen-specific Th I-type response.
The immunological mechanisms involved in maintenance of an asymptomatic microfilaremic state (MF) in patients with lymphatic filariasis remain undefined. MF patients have impaired filarial antigen (Ag)-specific lymphocyte proliferation and decreased frequencies (Fo) of Ag-specific T cells, and yet elevated serum IgE and antifilarial IgG4. To investigate the mechanism of Ag-specific anergy in MF patients in contrast to amicrofilaremic individuals with chronic lymphatic obstruction (CP), the Fo of Ag-specific lymphocytes from peripheral blood mononuclear cells secreting either IL-4 or IFN-gamma were assessed by filter spot enzyme-linked immunosorbent assay, and IL-10 and transforming growth factor-beta (TGF-beta) mRNA transcript levels were assessed by a semiquantitative reverse transcriptase polymerase chain reaction technique. The Fo of filaria-specific IL-4-secreting lymphocytes were equivalent in both MF (geometric mean [GM] = 1:11,700) and CP (GM = 1:29,300 P = 0.08), whereas the Fo of IFN-gamma-secreting lymphocytes were lower in MF (GM = 1:39,300) than in CP (GM = 1:4,200, P < 0.01). When the ratio of IL-4/IFN-gamma (T helper type 2 [Th2]/Th1)-secreting cells was examined, MF subjects showed a predominant Th2 response (8:1) compared with a Th1 response in CP individuals (1:4). mRNA transcript levels of IL-10 were also significantly elevated in MF compared with CP individuals (P < 0.01). Further, IL-10 and TGF-beta were shown to have a role in modulating the Ag-specific anergy among MF subjects, in that neutralizing anti-IL-10 or anti-TGF-beta significantly enhanced lymphocyte proliferation response (by 220-1,300%) to filarial Ags in MF individuals. These findings demonstrate that MF subjects respond to parasite antigen by producing a set of suppressive cytokines that may facilitate persistence of the parasite within humans while producing little clinical disease.
Lymphatic filariasis is a chronic disease characterized by a pronounced Th2 bias in the immune response and impaired antigen (Ag)-specific Th1 responses. We have used a mouse model of filariasis to investigate the role of the infective form (the third-stage larvae [L3]) in modulating the immune response. Subcutaneous infection of BALB/c mice with L3 of Brugia pahangi has a profound effect on Th cell function. By day 12 post-infection, spleen cells from these mice exhibited a dramatic reduction in concanavalin A-driven proliferation and interleukin-2 (IL-2) and gamma interferon (IFN-gamma) secretion in comparison with uninfected controls. However, exposure to L3 did not render the mice completely unresponsive; these animals mounted a strong Th2 response to the parasite, characterized by elevated levels of IL-4, IL-5, and IL-10 and parasite-specific serum immunoglobulin G (IgG), IgG1, and IgE. Treatment of spleen cells from L3-infected mice with neutralizing anti-IL-4 or recombinant IL-2 resulted in a dramatic increase in concanavalin A-induced proliferation and IL-2 and IFN-gamma production. Despite their defective polyclonal Th1 response, cells from L3 infected mice proliferated when stimulated with Ag, and this response was blocked by anti-IL-4. However, anti-IL-4 treatment failed to induce Ag-specific IL-2 or IFN-gamma production, indicating that B.pahangi-primed Th1 cells do not appear to be present or are still unable to respond even in the absence of IL-4.
To characterize immune responses associated with the putatively immune state in bancroftian filariasis (that is, both microfilaria and antigen free), humoral and cellular responses were compared among antigen- and microfilaria-negative, antigen-positive and microfilaria-negative, and microfilaria-positive individuals. Antifilarial isotype levels were measured by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cell responses were measured by proliferation, by bioassay for interleukin-2 (IL-2) and IL-10, and by reverse transcription-PCR for IL-4, IL-5, and gamma interferon. The absence of circulating filarial antigen was associated with Th1-like responses, including significantly higher proliferative (P < 0.001) and IL-2 (P = 0.008) responses and a higher prevalence of gamma interferon (0.02 < P < 0.1) responses. Significantly elevated antifilarial immunoglobulin G4 (IgG4) levels (P = 0.0035) were associated with antigenemia, whereas microfilaremia was associated with significantly decreased antifilarial IgG2 levels (P = 0.0014). IL-4 mRNA levels were not significantly different among the three groups; however, there was a subpopulation of microfilaremic individuals who did not make detectable levels of IL-4 mRNA and who produced low antifilarial IgG4 levels compared with those of individuals who had detectable levels of IL-4 mRNA. IL-5 mRNA levels also were not significantly different among groups; however, more microfilaremic individuals produced IL-5 mRNA in response to adult filarial antigens, and total parasite-specific IL-4 and IL-5 mRNA levels were significantly correlated (P = 0.05). Although longitudinal data are not currently available, the elevated Th1-like responses in antigen- and microfilaria-negative individuals are consistent with the hypothesis that these responses contribute to protection in putatively immune individuals.
The initial immune response to Schistosoma mansoni eggs presumably results in IL-4 production, as schistosome eggs are strong Th2-inducing antigens and the differentiation of antigen-specific Th2 cells is largely dependent on the presence of IL-4 during priming of naive Th cells. Consistent with this concept, intraperitoneal injection of mice with schistosome eggs results in an upregulation of IL-4 production by peritoneal exudate cells (PECs) within 12 h. Egg-induced IL-4 is rapidly bound by its receptor, suggesting that this cytokine is utilized by a cell type present at the site of antigen deposition or is complexed to soluble receptor. The peak of early IL-4 production is accompanied by a local eosinophilia and the apparent disappearance of mast cells. Studies utilizing either IL-4, IL-5, or mast cell-deficient mice indicate that the eosinophilia is dependent on mast cells and IL-5 and independent of IL-4. Strikingly, egg-induced IL-4 production is absent in animals lacking the early peritoneal eosinophilia. Immunocytochemical analysis of PEC following egg injection indicates that the eosinophils themselves make IL-4. These data strongly suggest that egg-induced IL-5 plays an essential role in recruiting eosinophils to the site of antigen deposition and that it is these eosinophils that then directly produce early IL-4.
To investigate the mechanisms of protective immunity operating in Loa loa infection, 56 persons from a L. loa–endemic village in southeast Gabon were examined over a 7-year period. The level of L. loa–specific IgG subclasses in defined parasitologic groups was compared by use of ELISA with either adult, microfilarial, or
third-stage larval (L3) antigens ofL. loa. With all antigen preparations, IgG1 levels were significantly higher in amicrofilaremic
persons than in persons with high or low levels of microfilariae. Moreover, there was a significant negative correlation between
IgG1 levels to L3 antigen and the density of microfilariae (Spearman's rs = –.701; P < .01). There was no correlation between density of microfilariae and levels of other IgG subclasses or of IgE. These data
indicate that IgG1 may playa role in the effector mechanism(s) involved in resistance against L. loa and suggest that L3 antigens may be important in eliciting protective responses.
Th2 lymphocyte responses under the control of IL-4 and IL-5 are frequently associated with protective responses to parasitic helminths. Studies on the role of these cytokines in acquired resistance to parasitic nematodes indicate that, in the case of gastrointestinal nematodes, immunity is mediated by IL-4, while immunity to tissue-dwelling nematodes is dependent on IL-5. Here we investigate the role of IL-5 and eosinophils in protective immunity to Onchocerca microfilariae in IL-4-deficient mice. In the absence of IL-4, and despite the up-regulation of Th1-type responses, immunity remains dependent on IL-5 and eosinophils. Protection was unaffected by the absence of Ab in B cell-deficient mice, confirming that IL-5 is not acting via either B cell differentiation, Ag presentation, or isotype switching mechanisms. These data demonstrate the dissociation of IL-4 and IL-5 in a functional model of protective immunity to a tissue dwelling nematode and cast doubt on the role of IL-4 in the generation of CD4+ T cell-mediated, IL-5-dependent immunity to Onchocerca microfilariae. Importantly, they also segregate T cell-mediated mechanisms of protective immunity from those characterized in ocular pathologic responses in onchocerciasis, which are dependent on IL-4.
Infection with the third-stage larvae (L3) of the filarial nematode Brugia results in a Th2-biased immune response in mice and humans. Previously we have shown that the production of interleukin 4 (IL-4) is critical for down-regulating polyclonal Th1 responses in L3-infected mice. However, the in vitro neutralization of IL-4 did not fully recover the defective polyclonal Th1 responses, nor did it result in the production of any antigen (Ag)-specific Th1 cytokines, suggesting that perhaps infection with L3 does not result in priming of Th1 cells in vivo. In this study, we analyzed the role of IL-10 and Ag-presenting cells (APCs) in the spleen as additional factors controlling the Th2 bias in infected mice. Our data show that IL-10 and APCs also contribute to the suppression of mitogen-driven Th1 responses of spleen cells from infected mice. In addition, the neutralization of IL-10 or the replacement of the resident APC population from spleen cell cultures resulted in the production of Ag-specific Th1 cytokines. Irradiated spleen cells from either L3-infected or uninfected mice were able to restore Ag-specific Th1 responses in vitro. Therefore, it appears that Brugia-reactive Th1 cells are primed following infection with L3, but are actively suppressed in vivo by a mechanism that involves IL-10 and the resident APC population, but not IL-4. These results indicate that a complex interplay of cytokines and cell populations underscores the Th2-polarized response in L3-infected mice.
Using a single vector targeting strategy, we have generated mice with a combined deficiency of interleukin (IL)-4 and IL-13 to clarify their roles in T helper type 2 (Th2) cell responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of the double-deficient mice with those generated by wild-type, IL-4-deficient, and IL-13-deficient mice. Using a pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that although eosinophil infiltration, immunoglobulin E, and IL-5 production are reduced in the IL-4-deficient mice and IL-13-deficient mice, they are abolished only in the combined absence of both cytokines. Furthermore, IL-4/13-deficient animals are severely impaired in their ability to expel the gastrointestinal nematode Nippostrongylus brasiliensis. Unexpectedly, N. brasiliensis-infected IL-4/13-deficient mice developed elevated IL-5 and eosinophilia, indicating that compensatory mechanisms exist for the expression of IL-5, although serum IgE remained undetectable. IL-4/13-deficient mice default to a Th1-like phenotype characterized by the expression of interferon gamma and the production of IgG2a and IgG2b. We conclude that IL-4 and IL-13 cooperate to initiate rapid Th2 cell-driven responses, and that although their functions overlap, they perform additive roles.
B7 costimulation is a required component of many type 2 immune responses, including allergy and protective immunity to many nematode parasites. This response includes elevations in Th2 cytokines and associated effector functions including elevations in serum IgG1 and IgE and parasite expulsion. In studies of mice infected with Trichuris muris, blocking B7 ligand interactions inhibited protective immunity, suppressed IL-4 production, and enhanced IFN-gamma production, but unexpectedly did not inhibit production of the Th2 cytokine, IL-13. Blocking both IFN-gamma and B7 restored protective immunity, which was IL-13 dependent, but did not restore IL-4 or associated IgE responses. Although IL-13 was required for worm expulsion in mice in which both IFN-gamma and B7 were blocked, IL-4 could mediate expulsion in the absence of both IL-13 and IFN-gamma. These studies demonstrate that 1) B7 costimulation is required to induce IL-4, but not IL-13 responses; 2) IL-13 is elevated in association with the IFN-gamma response that occurs following inhibition of B7 interactions, but can only mediate IL-4-independent protection when IFN-gamma is also inhibited; and 3) increased IL-13 production, in the absence of increased IL-4 production, is not associated with an IgE response, even in the absence of IFN-gamma.
We have investigated the roles of gamma interferon (IFN-γ) and interleukin-4 (IL-4) in host defense against Brugia malayi. Our data suggest that the lack of either IFN-γ or IL-4 prolongs the time required to achieve sterile immunity, suggesting
that both canonical type 1 and type 2 responses are involved in the clearance of infection.
The study of protective immune mechanisms effective against filarial nematodes has been hampered by the inability of these
important human pathogens to infect laboratory mice. Recently,Litomosoides sigmodontis, a natural parasite of rats, has been developed as a valuable model for the study of filarial infection. BALB/c mice are
fully susceptible to infection with L. sigmodontis third-stage larvae and develop patent infection. In contrast, mice on the C57BL background are resistant, and parasites undergo
only a single molt and do not mature to adulthood. We used interleukin-5 (IL-5)-deficient mice on the C57BL/6 background to
address the role of IL-5 and eosinophils in the innate resistance of C57BL/6 mice. We found no differences in parasite survival
between IL-5-deficient and C57BL/6 mice. However, when these mice were used for the analysis of vaccine-mediated immunity,
a critical role for IL-5 was elucidated. Mice genetically deficient in IL-5 were unable to generate a protective immune response
when vaccinated with irradiated larvae, whereas C57BL/6 mice were fully protected from challenge infection. These studies
help to clarify the highly controversial role of eosinophils in filarial infection.
Although exogeneous "danger" signals such as LPS can activate APC to produce a Th1 response, the nature of events initiating a Th2 response is controversial. We now show that pathogen-derived products have the capacity to induce bone marrow-derived dendritic cell cultures to acquire a phenotype that promotes the differentiation of naive CD4+ T cells toward either a Th1 or Th2 phenotype. Thus, LPS-matured dendritic cells (DC1) promote a Th1 response (increased generation of IFN-gamma and reduced production of IL-4) by Ag-stimulated CD4+ T cells from the DO.11.10 transgenic mouse expressing a TCR specific for an OVA peptide (OVA323-339). In contrast, a phosphorylcholine-containing glycoprotein, ES-62, secreted by the filarial nematode, Acanthocheilonema viteae, which generates a Th2 Ab response in vivo, is found to induce the maturation of dendritic cells (DC2) with the capacity to induce Th2 responses (increased IL-4 and decreased IFN-gamma). In addition, we show that the switch to either Th1 or Th2 responses is not effected by differential regulation through CD80 or CD86 and that a Th2 response is achieved in the presence of IL-12.
Infection of BALB/c mice with microfilariae (mf) of Brugia pahangi leads to the suppression of antigen (Ag)-specific proliferative responses in the spleen. The proliferative defect is dependent
on inducible nitric oxide synthase (iNOS) activity, since inhibition of iNOS with either l-N-monomethyl arginine (l-NMMA) or aminoguanidine reversed defective proliferation. Splenocytes from mf-infected animals produce high levels of gamma
interferon (IFN-γ) upon in vitro restimulation with Ag, and experiments in IFN-γ receptor-deficient (IFN-γR−/−) mice demonstrated that signaling via the IFN-γR is essential in the induction of NO production and subsequent proliferative
suppression. Restimulation of splenocytes from mf-infected animals with an extract of Acanthocheilonema viteae, a related filarial worm which lacks endosymbiotic bacteria, also resulted in NO production and proliferative suppression,
demonstrating that lipopolysaccharide of bacterial origin is not essential to the induction of iNOS activity. These results
extend previous observations that infection with different life cycle stages of Brugia leads to the development of differentially polarized immune responses and demonstrate one method by which these differences
may exert their effects on the proliferative potential of cells from infected animals.
BALB/c mice immunized with radiation attenuated third stage larvae of the filarial nematode Brugia pahangi are strongly immune to challenge infection. Investigation of the profile of cytokines secreted by spleen cells from immune mice stimulated in vitro with either parasite Ag or with Con A revealed high levels of IL-5 and IL-9 and moderate levels of IL-4. In contrast, secretion of IFN-gamma by spleen cells from immune animals was negligible. Spleen cells from control mice secreted low levels of all cytokines assayed. Levels of parasite-specific IgE were significantly elevated in immune animals and a peripheral blood eosinophilia was observed, which exhibited a biphasic distribution. Our results are consistent with the preferential expansion of Th2 cells in immune animals and provide the basis for dissecting the means by which radiation attenuated larvae of filarial nematodes stimulate immunity.
Infection with the third-stage larvae (L3) of the filarial nematode Brugia results in a Th2-biased immune response in mice and humans. Previously we have shown that the production of interleukin 4 (IL-4) is critical for down-regulating polyclonal Th1 responses in L3-infected mice. However, the in vitro neutralization of IL-4 did not fully recover the defective polyclonal Th1 responses, nor did it result in the production of any antigen (Ag)-specific Th1 cytokines, suggesting that perhaps infection with L3 does not result in priming of Th1 cells in vivo. In this study, we analyzed the role of IL-10 and Ag-presenting cells (APCs) in the spleen as additional factors controlling the Th2 bias in infected mice. Our data show that IL-10 and APCs also contribute to the suppression of mitogen-driven Th1 responses of spleen cells from infected mice. In addition, the neutralization of IL-10 or the replacement of the resident APC population from spleen cell cultures resulted in the production of Ag-specific Th1 cytokines. Irradiated spleen cells from either L3-infected or uninfected mice were able to restore Ag-specific Th1 responses in vitro. Therefore, it appears that Brugia -reactive Th1 cells are primed following infection with L3, but are actively suppressed in vivo by a mechanism that involves IL-10 and the resident APC population, but not IL-4. These results indicate that a complex interplay of cytokines and cell populations underscores the Th2-polarized response in L3-infected mice.
This study reports reversals in microfilarial density and the accompanying changes in cellular immune responses to filarial antigens of 39 individuals (11 microfilaria-positives, 22 microfilaria-negatives and six converters) living in an area endemic for brugian filariasis. Microfilarial counts decreased from April, the end of the rainy season to July, middle of the dry season (g.m. 88 mf/ml and 38 mf/ml, respectively; P = 0.001) and subsequently increased in November, the beginning of the rainy season (P = 0.088). Whereas the proliferative responses remained low throughout the study period in microfilaraemic individuals, in amicrofilaraemics these responses changed in the opposite direction to that of microfilarial densities. In three converters, proliferation changed in the opposite direction to the presence or absence of microfilariae. Cytokine analysis in the converters revealed that interferon- was most affected by the shifts in microfilarial densities. In contrast, interleukin-4 responses showed little correlation with changes in parasite densities.
Embryonic stem (ES) cell lines have been derived from the inner cell mass of day 3.5 blastocysts of the inbred mouse strain BALB/cJ. Twenty-three lines were karyotyped and three were selected for injection into C57BL/6J host blastocysts. Two of the three lines, BALB/c-I and BALB/c-IV, produced germ-line chimaeras. The suitability of the BALB/c-I line for gene targeting experiments was tested by transfecting a targeting construct for the interleukin-4 (IL-4) gene. Transfected BALB/c-I cells exhibited efficient homologous recombination of the targeting vector and transmitted the induced mutation through the germline. This newly-characterized BALB/c-ES cell line thus provides an alternative to the traditional 129-derived and the recently described C57BL/6 embryonic stem cell lines, and will be useful in disrupting genes involved in the immune system. Furthermore, the genetically pure BALB/c IL-4 deficient mice will aid in studying the role of IL-4 in several infectious disease models in which the BALB/c mouse is a susceptible strain.
Mice in which either the IL-4 or the IL-13 gene has been disrupted (IL-4 KO and IL-13 KO) were susceptible to infection with the intestinal nematode Trichuris muris, whereas their wild-type littermates were highly resistant and expelled the parasite. IL-4 KO mice showed diminished Th2-type responses with T. muris infection and also failed to produce parasite-specific IgG1 Abs. Although IL-13 KO mice made reduced Th2-type responses early in infection, they were capable of generating strong Th2-type responses at later time points and were unable to regulate the magnitude of their Ab isotype response. These results confirm the importance of IL-4 in resistance to T. muris and provide the first demonstration of an important role for IL-13 in resistance to helminth infection. The IL-13 KO mouse had a separate phenotype to that of the IL-4 KO mouse, suggesting that both IL-4 and IL-13 play important yet different roles in mediating immunity to intestinal helminths.
Triatomine species influenced volume of feces produced; Triatoma dimidiata produced the largest volume of feces followed by Panstrongylus megistus, Rhodnius prolixus, and Triatoma infestans. Moreover, stage and sex affected fecal production; 5th-stage female nymphs excreted the largest volume of feces. The amount of blood ingested was significantly correlated with the volume of feces produced in 10 out of 11 experimental groups. Triatome size and volume of feces produced were less closely correlated. Indeed, a "threshold" minimum amount of blood must be ingested before bugs are stimulated to defecate. The defecation habits of triatomines probably influence the vectorial capacity of a triatomine species to a lesser degree than do the density of domestic infestations, host affinity, and the degree of adaptation to the domestic habitat.
The susceptibility of several strains of inbred mice to infection with the filarial worm Brugia pahangi has been examined. BALB/C, C57BL/10, C3H/He, 101, CBA/Ca mice, congenitally asplenic (DH/+) mice and their normal litter-mates (+/+) were each challenged by the intraperitoneal inoculation of 50 infective larvae. During the first four weeks of infection high (19-42%) larval recoveries were obtained from the CBA/Ca, BALB/C and Dh/+ mice but fewer than 10% of inoculated larvae were recovered from C3H/He, 101; C57BL/10 and +/+ mice. Larval growth rates in all mice were similar. BALB/C and Dh/+ mice only were examined later than four weeks after infection. The yield of adult worms from BALB/C was 7.5% at 16 weeks and from Dh/+ 4.2% at 21 weeks. Microfilariae were present in the peritoneal fluids but not the blood of some mice harbouring both adult male and female worms.
Resistance of intact mice to infection with the filarial nematode, Brugia malayi is dependent on the presence of T cells. To investigate the role of Th2 cells in this protection, mice with a targeted disruption of the IL-4 gene were infected with different developmental stages of B. malayi. We examined the phenotypic changes in the immune response and the survival of each stage in these mice. In wild-type mice, adult female worms induce Th2 responses, characterized by antigen-specific IgG1 production, elevated IgE, and marked IL-4 secretion by splenocytes stimulated in vitro with Brugia extract. However, first stage larvae (microfilariae), induce Th1 responses with the appearance of antigen-specific IgG2a, IgG2b, and IgG3 and IFN-gamma secretion by splenocytes. Infection of IL-4-deficient mice revealed a dramatic change in the response to adult worms, with a severe reduction in IgG1 production and a corresponding increase in the production of IgG2a, IgG2b, IgG3, and IFN-gamma release. The switch to Th1-type responses was particularly marked in IL-4-deficient recipients of female worms, which continually release live microfilariae. In the absence of IL-4, down-regulation of the microfilarial-induced Th1 response does not occur. Despite these profound alterations to the immune response in IL-4-deficient mice, survival of infective larvae, adult worms, or microfilariae in the peritoneal cavity was unaffected. In mice, therefore, the prominent Th2-type response elicited by filarial parasites may not be an essential component of the host protective immune response.
Natural infection with filarial nematode parasites shows many characteristics of a Th2 immune response. In these infections, long-lived adult worms inhabit the lymphatics, releasing laval microfilariae (Mf) into the blood stream. To compare the effect of these different developmental stages on the mammalian immune system, Mf and adult worms of either sex were implanted into BALB/c mice, in which they survive for at least 28 days. Serum Ab responses showed that whereas Mf stimulated specific Abs of all IgG subclasses, but little total IgE, adult worms stimulated only IgG1 and IgE responses. Splenocytes from implanted mice were stimulated in vitro with specific Ag or Con A and assayed for proliferation and profiles of cytokine secretion. Cells from Mf-infected mice secreted high levels of IFN-gamma (30 U/ml) throughout infection, but very little IL-4 at the early time points. By day 28 postinfection, however, splenocytes from Mf-infected mice showed some IL-4 secretion in response to specific Ag (40 U/ml). The IFN-gamma response to Mf was found to be independent of the inoculum dose in the range of 10(2) to 10(6) organisms. In contrast, splenocytes taken from adult worm-implanted mice on days 14, 21, and 28 postinfection produced high levels of IL-4 (up to 435 U/ml) and negligible amounts of IFN-gamma despite the production of large numbers of Mf by adult female worms. CD4+ cells were primarily responsible for this IL-4 production. These results demonstrate that adult filarial parasites, and females in particular, exert a rapid polarization of the immune response in a Th2-like direction, but that this effect may be modulated by the Mf stage.
A significant reduction in challenge worm survival occurred when BALB/cBYJ mice were vaccinated against Onchocerca volvulus infective third stage larvae (L3) by using irradiated O. volvulus L3. Challenge infections consisted of L3 implanted in diffusion chambers, which were used as a means to contain, and thus efficiently recover, the larvae from the host. The goal of the present study was to describe the mechanism of immune-mediated killing of O. volvulus L3 in diffusion chambers in mice. Direct contact between host cells and parasites was required for killing of larvae in immunized hosts. To define the mechanism of immune-mediated killing in this system, the time of influx of cells and cytokines into the infection site was compared with the time challenge infections were killed. The only cell type that was found to increase in diffusion chambers in immunized mice was eosinophils; maximal levels of eosinophils were coincident with the time of parasite killing. IL-5 was found in diffusion chambers of immunized mice coincident with the time of parasite killing; IL-5 was not found in diffusion chambers recovered from control mice. Significant levels of IFN-gamma were absent in the diffusion chambers of both groups. Immunized mice were treated with mAb to eliminate IL-5 or IL-4 to assess the role these cytokines or their by-products play in larval killing. Elimination of either IL-5 or IL-4 significantly reduced the protective effects of vaccination against larval O. volvulus.
In the three years since its discovery, the pleiotropic cytokine interleukin-10 (IL-10) has been implicated as an important regulator of the functions of lymphoid and myeloid cells. IL-10's ability to block activation of cytokine synthesis and several accessory cell functions of macrophage renders this cytokine a potent suppressor of the effector functions of macrophages, T cells, and NK cells. In addition, IL-10 likely contributes to regulating proliferation and differentiation of B cells, mast cells, and thymocytes. The Epstein-Barr virus genome encodes a homolog of IL-10 (BCFR1, viral IL-10, vIL-10) which shares many of the cellular cytokine's biological activities and may therefore play a role in the host-virus interaction. This article reviews current studies of IL-10's biological activities and discusses its possible roles in regulation of immune responses.
The effect of irradiation on the third stage larvae of the filarial nematode Brugia pahangi was investigated. Labelling with 35S methionine of control or irradiated L3, post-infective L3 or L4 revealed no consistent alterations in the pattern of proteins synthesized. The only significant difference observed was in 125I labelling, where the specific activity of labelling of soluble cuticular proteins was lower in irradiated than in control parasites. This difference may be related to the reduced size of irradiated parasites rather than to a specific effect of irradiation on the expression of cuticular proteins. Irradiated parasites recovered on day 14 post-infection were significantly shorter than control parasites. Irradiation also appeared to have a lethal effect on male parasites, as no recognizable males were recovered from animals given irradiated L3, nor were microfilariae ever observed in these animals. The mechanisms by which irradiation may enhance the immunogenicity of L3 of filarial nematodes are discussed.
BALB/c mice immunized with radiation attenuated third stage larvae of the filarial nematode Brugia pahangi are strongly immune to challenge infection. Investigation of the profile of cytokines secreted by spleen cells from immune mice stimulated in vitro with either parasite Ag or with Con A revealed high levels of IL-5 and IL-9 and moderate levels of IL-4. In contrast, secretion of IFN-gamma by spleen cells from immune animals was negligible. Spleen cells from control mice secreted low levels of all cytokines assayed. Levels of parasite-specific IgE were significantly elevated in immune animals and a peripheral blood eosinophilia was observed, which exhibited a biphasic distribution. Our results are consistent with the preferential expansion of Th2 cells in immune animals and provide the basis for dissecting the means by which radiation attenuated larvae of filarial nematodes stimulate immunity.
We have shown previously that immunisation of mice and pigs with recombinant 26 kDa GST (reSjc26GST) induces a pronounced anti-fecundity effect after experimental infection with Chinese Schistosoma japonicum. We report here that anti-fecundity immunity can also be induced against reSjc26GST in Chinese water buffaloes (Bos buffelus), important reservoir hosts for S. japonicum in China. Anti-Sjc26GST antibodies were produced in immunised buffaloes and, following challenge with S. japonicum cercariae, a 22.3% reduction in worm numbers was evident in vaccinated when compared with control animals. The anti-fecundity effect was characterised by a significant decrease in faecal egg output and eggs deposited in host tissues with those in the liver and intestine being reduced by about 50%. In addition to the anti-fecundity effect, reSjc26GST reduced by nearly 40% the egg-hatching capacity of S. japonicum eggs into viable miracidia. In terms of vaccination strategy, these effects would combine to diminish pathology in animals immunised with reSjc26GST and reduce transmission of schistosomiasis japonica.
Lymphatic filariasis in man is characterized by a profound bias in the immune response. Parasite-specific Th1 responses are dramatically down-regulated while Th2 responses dominate. We have used the infective larval stage of the nematode parasite Brugia pahangi, a potent Th2 inducer in naive mice, to examine cytokine production during the initiation phase of the response. For comparative purposes, the early cytokine transcription pattern elicited by microfilariae (mf), another life cycle stage of the parasite known to induce a primary Th1 response, was analysed in parallel. At 24 h post-infection (p.i.) a burst of IL-4 transcription was detected in the draining popliteal lymph node of L3-infected animals. IL-4 was the only cytokine transcript detectable at this early time point and was not present in mf-infected mice. From day 4 p.i. onwards, the L3 elicited a Th2 response as defined at the level of cytokine mRNA and protein production by CD4+ cells. In contrast, mf stimulate high levels of IFN-gamma mRNA at day 4 p.i. in the absence of IL-4 or IL-10 induction. Cell selection analysis indicated that IL-4 produced at 24 h derived from a population of CD4-CD8- alphabeta T cells. These results suggest that triggering of an unusual double-negative T cell population to secrete IL-4 at the very outset of infection with the L3 of B. pahangi may be the critical factor favouring the development of antigen-specific Th2 cells in response to this stage of the parasite.
C57BL/6 mice genetically deficient in interleukin-5 (IL-5-/-) and normal C57BL/6 (IL-5+/+) mice were infected with larvae of a homogonic strain of the nematode Strongyloides ratti. In primary infections both male and female IL-5-/- mice released two to four times more eggs and larvae than IL-5+/+ mice. IL-5-/- mice harboured about 60% more intestinal worms, which were more fecund, than IL-5+/+ mice. The duration of the infection was similar in normal and IL-5-deficient mice. Both IL-5-/- and IL-5+/+ mice resisted a secondary infection. IL5-/- mice lost more weight during the infection than normal mice and took longer to regain their initial weight after expelling the worms. The number of eosinophils increased in the bone marrow, peritoneal cavity and small intestine of IL-5+/+ mice, but not IL-5-/- mice, following infection. No significant differences between infected IL-5+/+ and IL-5-/- mice in mast cells or other leucocytes were observed in the peritoneal cavity. Thus, IL-5 functions to protect the host in a primary infection of S. ratti by limiting the number and fecundity of worms establishing in the small intestine. This protection is correlated with elevated blood and tissue eosinophilia which occurs in normal mice but not in IL-5-/- mice.
In Wuchereria bancrofti transmission areas, three groups of individuals have been identified, according to the presence or absence of microfilariae or adult worm derived molecules in the blood compartment. These groups likely reflect individuals with different permissivity/resistance to the complete development of W. bancrofti cycle. The profile of filarial-specific immunoglobulins was analysed in W. bancrofti-exposed individuals in French Polynesia, according to the presence or absence of microfilariae (Mf) and adult worms, measured by Og4C3 circulating antigen. Individuals harbouring adult worms, have higher filarial-specific IgG4 but lower IgG3 and IgE levels, than adult worm-free individuals, independently of the presence of Mf. Low filarial-specific IgG1 and IgG2 levels were associated with the presence of Mf but independent of the presence/absence of adult worms. The filarial antibody responses were associated with the parasitological status of individuals but not with clinical symptoms such as hydroceles or limb lymphangitis or elephantiasis. The reduction of filarial-specific immunoglobulin levels was higher after treatment with diethylcarbamazine, than ivermectin, which likely reflects the better effect of the former on W. bancrofti adult worms. However, reduction of antibody levels was also observed in Mf-and adult worm-negative individuals. This could be due to the overall reduction of W. bancrofti transmission in the island where this study took place.
Rajan, T. V., Babu, S., Sardinha, D., Smith, H., Ganley, L., Pacior kowski, N., and Porte, P. 1999. Life and death of Brugia malayi in the mammalian host: passive death vs active killing. Experimental Parasitology93, 120–122.
In this study we characterized Th2 responses in the absence of IL-4. We show that ST2L, a stable Th2 marker, is expressed at similar levels in Leishmania major-infected IL-4-deficient (IL-4(-/-)) and wild-type BALB/c (IL-4(+/+)) mice. Th2 cytokines are secreted by in vivo differentiated lymphocytes in response to specific activation in the absence of IL-4. Although IL-13 is produced, its neutralization did not alter the outcome of infection. Thus, we demonstrate that Th2 differentiation as assessed by the expression of ST2L and the production of Th2 cytokines can occur in vivo in the absence of IL-4.
A key feature of nematode infection is a bias towards a type 2 immune response. To investigate the role that antigen-presenting cells (APC) may play in promoting this bias, we used adherent peritoneal exudate cells (PEC) recruited in response to the filarial nematode Brugia malayi, to stimulate naïve T cells from pigeon cytochrome c (PCC)-specific TCR transgenic (PCC-tg) mice. Although the proliferation of PCC-tg T cells was inhibited by parasite- induced PEC during primary stimulation, they proliferated normally upon secondary stimulation and were not rendered anergic. However, PCC-tg T cells primed by suppressive APC differentiated into IL-4-producing Th2 cells upon secondary stimulation instead of IFN-gamma-producing Th1 cells, as has been previously described. Studies with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells indicated that Th2 differentiation was associated with the inhibition of (or failure to stimulate) IFN-gamma production during primary stimulation. Interestingly, blocking antibodies against TGF-beta (but not IL-10) restored the differentiation of IFN-gamma-producing Th1 cells. Identical results with CFSE-labeled cells were obtained using purified IL-4-dependent F4/80(+) macrophages. These data indicate that T cells exposed to parasite-induced alternatively activated macrophages are driven towards Th2 differentiation. This may be an important factor in the Th2 bias that accompanies nematode infection.
Human filarial infection presents a spectrum of clinical states with two major poles: asymptomatic microfilaraemia and amicrofilaraemic chronic disease. Microfilaremia is associated with a Th1-type tolerance, and maximal IgG4 antibodies, while elephantiasis patients react across a broad range of immune parameters. In this review, Rick Maizels and his colleagues discuss recent advances in the immunology of human filariasis and present a summary of their latest studies in an endemic area of Indonesia.
NO contributes to proliferative suppression in a mouse model of filariasis
- O Connor
- R Jenson
- J Devaney