Neospora caninum: a cause of immune-mediated failure of pregnancy?

Opinion

TRENDS in Parasitology Vol.18 No.9 September 2002

Neospora caninum: a cause of immunemediated failure of pregnancy? Helen E. Quinn, John T. Ellis and Nicholas C. Smith Resistance to many intracellular protozoan parasites is dependent on T helper cell 1 cytokine responses. This has important repercussions for pregnant females because strong T helper cell 1 cytokine responses are incompatible with successful pregnancy. Thus, there are two possible consequences of infection with protozoans such as Leishmania major, Plasmodium falciparum and Toxoplasma gondii during pregnancy: (1) pregnancy is compromised; or (2) resistance to the parasite is compromised. The apicomplexan Neospora caninum is a parasite renowned for its association with abortion in cattle. Furthermore, a major route of transmission for this parasite is congenital. The evidence for the hypothesis that T helper cell 1 cytokines play a role in these events is reviewed here. Published online: 6 August 2002

Helen E. Quinn John T. Ellis* Dept of Cell and Molecular Biology, University of Technology, Sydney, PO Box 123, Broadway, New South Wales 2007, Australia. *e-mail: john.ellis@ uts.edu.au Nicholas C. Smith Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, PO Box 123, Broadway, NSW, 2007, Australia.

Many parasites induce polarized immune responses by their hosts. These responses are characterized by potentially dichotomized cytokine profiles. At one extreme are relatively large, multicellular helminth parasites of extracellular spaces, which elicit production of T helper cell (Th) 2 cytokines [e.g. interleukin (IL)-4, IL-5, IL-6, IL-9, IL-10 and IL-13] and favour (in mice at least) the generation of immunoglobulin (Ig) G1 and IgE antibodies [1]. At the other extreme are intracellular protozoan parasites, which stimulate Th1 cytokine responses dominated by cytokines such as IL-12, interferon (IFN) γ and tumour necrosis factor (TNF) α, leading to activation of pathways that generate free oxygen radicals (FOR), and nitric oxide (NO) and its metabolites, among other factors which are potentially lethal for many protozoa [2]. Th1 responses in mice are classically associated with antibodies of the IgG2a isotype plus, potentially, IgG2b and IgG3 isotypes, and inhibition of IgG1, IgE and IgM [3]. Cytokines and pregnancy

Cytokine profiles also have physiological implications, perhaps the most profound being the detrimental affect of Th1 cytokines, and associated cells and mediators, on pregnancy. Thus, natural killer (NK) cells, IFN-γ, TNF-α and NO can be deleterious to the well-being of the foetoplacental unit, and can be capable of inducing abortion and/or foetal resorption [4]. Hence, the Th2 cytokine profiles http://parasites.trends.com

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are the default option during pregnancy, with foetoplacental tissues producing cytokines such as IL-4, IL-5 and IL-10. This profile may be the result of the high levels of progesterone maintained during pregnancy because progesterone promotes Th2 proliferation [5]. Progesterone is also known to inhibit NO production, TNF-α production and NK cell activity [6–8]. Ref. [9] presents a comprehensive review of the effects of sex- and pregnancy-associated hormones on immunity to protozoan infections. The effects of this local Th2 cytokine bias in foetoplacental tissues may be felt more systemically and have important repercussions for immune responsiveness to infection during pregnancy; this has been exemplified using Leishmania major infection of pregnant mice [10,11] and is summarized in Fig. 1. Leishmania major and pregnancy

Leishmania major infection in mice is the quintessential illustration of the importance of a Th1 response for the control of intracellular protozoan infections; this model has been investigated exhaustively [12,13]. Briefly, certain inbred strains of mice (e.g. C3H and C57BL/6) mount a strong Th1 response to L. major, which controls the parasite, whereas other inbred strains (most notably BALB/c) mount strong Th2 responses to the parasite and are not capable of resolving infection. Resistant mice produce IL-12 within hours of infection with L. major. The origins of this IL-12 have not been established, but seem most likely to be produced from antigen-presenting cells, either macrophages or dendritic cells. IL-12 activates NK cells, which are the early source of IFN-γ generated in response to L. major. Subsequently, TNF-α, FOR and NO all play a role in controlling the spread or even eliminating the parasite. Thus, a range of cells and factors implicated in failed gestation are crucial for resistance to L. major.

Th1 response

Non-viable pregnancy

Inflammatory cytokines

Infection controlled

Maternal immunity

Th2 response

Pro-gestation cytokines

Infection may not be controlled Viable pregnancy TRENDS in Parasitology

Fig. 1. A dominant T helper cell (Th) 1 response in the maternal immune system during pregnancy could result in the overproduction of inflammatory cytokines. These allow the control of parasitic protozoan infections, but are deleterious to the survival of the foetus. Conversely, if a dominant Th2 response occurs, pro-gestation cytokines will be produced, which will support the development of the foetus. However, these cytokines may not adequately control a parasitic infection, causing an increased parasite burden in the mother.

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The combination of pregnancy and L. major infection in resistant mice may be predicted to have two outcomes: (1) pregnancy may compromise resistance to the parasite; and (2) a strong Th1 response to the parasite may compromise pregnancy. In fact, both of these outcomes occur, and pregnant C57BL/6 mice effectively display two phenotypes. In the first phenotype, parasite burden is increased when compared with non-pregnant infected control mice, in association with enhanced expression of cytokines such as IL-4, IL-5 and IL-10, and reduced production of IFN-γ by lymph and spleen cells [10]. IgG1 dominates the antibody profile in these pregnant mice, whereas non-pregnant infected control mice display elevated levels of IgG2a. In the second phenotype, the frequency of viable pregnancies in infected mice is much less than in pregnant non-infected mice. This reduced success rate of gestation may be the result of either increased implantation failure or foetal resorption, depending on the time of infection relative to conception but, in both cases, this corresponds with a relatively low placental production of IL-4 and IL-10, and an increase in IFN-γ and TNF-α production by placental cells [11]. Apicomplexan parasites and pregnancy

Infection with apicomplexan parasites has long been recognized to have adverse effects on pregnancy [14]. Thus, foetal loss or low-birth-weight babies are frequent effects in pregnant women infected with Plasmodium falciparum and, furthermore, pregnant women suffer higher incidences of infection, more severe pathology and higher mortality than do any other cohort of the population, except perhaps for children under five years of age [15]. It has been hypothesized that many of the manifestations of malaria in pregnancy can be explained on the basis of the Th1–Th2 cytokine dichotomy [16] and this notion has been supported by recent field data. Studies of women from Kenya and from Malawi exposed to malaria during pregnancy indicated that increased TNF-α expression in placental cells is associated with low-birth-weight in infants [17,18]. The Kenyan study also identified an increase in IFN-γ expression and a decrease in IL-10 expression in placental cells as factors leading to low-birth-weight of infants born to Plasmodium-infected mothers [17]. Furthermore, among primigravid women, placental TNF-α concentrations are significantly elevated in those women suffering severe anaemia [17]. Toxoplasma gondii also has an adverse effect on pregnancy, but the timing of infection relative to gestation is a crucial factor. Thus, chronically infected pregnant humans, sheep or mice harbouring tissue cysts generally do not transmit parasites congenitally nor suffer any ill effects themselves [9]. The exception to this appears to be in chronically infected outbred and field mice, which can transmit parasites congenitally [19]. However, if infection occurs in the http://parasites.trends.com

first trimester, the risk of abortion is relatively high, although transmission of parasites to the foetus is low. By contrast, if infection occurs during the third trimester, the risk of abortion is relatively low, whereas congenital transmission of T. gondii is relatively frequent. Roberts et al. [9] argue that this may be explained on the basis of hormone levels and cytokine profiles. Thus, in the first trimester, levels of pregnancy hormones, such as progesterone, are relatively low so there is no or little Th2 cytokine polarization and every chance that the mother will mount a Th1 response to deal with the parasite – in the third trimester, hormone levels are high, the Th2 bias is firmly established; hence, control of the parasite is compromised and congenital transmission occurs. This is supported by evidence from murine studies. For example, pregnant mice are more susceptible to infection with T. gondii and this is associated with a reduced ability to produce IFN-γ [20]. Furthermore, administration of Th1 cytokines, such as IFN-γ and IL-2, reduces mortality in pregnant mice infected with T. gondii [21]. The immune response of mice to T. gondii during pregnancy has also been studied using transgenic IL-4-deficient mice; pregnant wild-type mice are more susceptible than IL-4-deficient mice to toxoplasmosis, showing increased parasite loads. Pregnant IL-4-deficient mice also demonstrate a decreased transmission rate to the foetus when compared with wild-type mice [22]. Neospora caninum

Neospora caninum has become of international concern owing to the connection of infection by this parasite with abortion in dairy and beef cattle, worldwide [23]. Thus, N. caninum demonstrates the deleterious affects on pregnancy typical of other apicomplexan parasites. Infected adult cattle do not exhibit clinical signs of disease and the only manifestation of infection is foetal loss and abortion. This leads to increased culling in a dairy herd, can reduce milk production and decreases the value of female breeding cattle [24]. Vertical transmission (from mother to foetus) is the major mode of transfer of this parasite in dairy cattle, with a high proportion of infected cows transmitting infection to their offspring [25–27]. At present, the precise mechanism of foetal loss and vertical transmission is poorly understood, but it seems likely that immune factors influence both. It has been demonstrated that by pretreating pregnant mice with a single dose of N. caninum tachyzoite crude lysate before infection, vertical transmission to offspring can be reduced [28]. This suggests a role for the immune system in preventing vertical transmission and it has also been shown that abortion in infected mice does not require the presence of the parasite in the placenta or foetal tissue; in a study in which mice were infected with N. caninum before or during pregnancy, no evidence of parasites was observed in foetal tissue, despite the high level of abortion recorded [29].

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TRENDS in Parasitology Vol.18 No.9 September 2002

Cytokines and immunity to Neospora caninum

Available evidence suggests that the immune response to N. caninum, similar to other intracellular protozoan parasites, is dominated by Th1 cytokines. Within 24 h of infection with N. caninum, spleen cells from A/J mice express significant levels of IL-12 messenger RNA (mRNA) and, after seven days, the cells express significant quantities of IFN-γ mRNA too [30]. These spleen cells proliferate in vitro when stimulated with N. caninum antigens. Neospora caninum infection also stimulates the production of IL-12 and IFN-γ by spleen cells from C57BL/6 mice, and IgG2a and IgG2b are the dominant antibody isotypes produced by these mice [31]. The importance of IFN-γ in parasite resistance has been demonstrated in a study comparing susceptibility to N. caninum in three strains of mice. The cytokine profile from spleen cells from infected mice showed a high IFN-γ:IL-4 ratio in resistant B10.D2 mice, whereas a low IFN-γ:IL-4 ratio was associated with susceptible BALB/c and C57BL/6 mice. The antibody profiles of these mice demonstrated predominantly IgG2a antibodies in resistant mice and IgG1 antibodies in susceptible mice [32]. The immune response to N. caninum has also been studied by blocking cytokines or by administering cytokines to mice during an infection. Neutralization of IL-12 and IFN-γ in vivo with antibodies increases susceptibility to infection [30]. Mice that have IFN-γ function blocked by treatment with an anti-IFN-γ antibody show an increase in mortality and parasite load, an increased ratio of antigen-specific IgG1:IgG2a antibodies and a decreased ratio of IFN-γ:IL-4. Mice treated with recombinant IL-12 show a decrease in brain lesions and parasite load, a decreased ratio of antigen-specific IgG1:IgG2a antibodies and an increased ratio of IFN-γ:IL-4. This IL-12 effect was demonstrated to be dependent on IFN-γ by concomitant neutralization of IFN-γ [33]. A similar observation was made in another study, in which IFN-γ antibody was administered to mice before and after infection with N. caninum. These mice died within 18 days of infection, whereas mice that had been given only parasites survived for >30 days [34]. NO is also important in murine resistance to N. caninum; mice deficient in inducible nitric oxide synthase (iNOS), the enzyme that produces NO, are more susceptible to N. caninum infection than wild-type mice [35]. There is only limited data on the immune response in cattle infected with N. caninum, but they suggest that, similar to mice, cattle mount a Th1 cytokine-dominated response to N. caninum. Cattle experimentally infected with N. caninum show proliferation of lymphocytes 4–6 days post inoculation (pi). Increased production of IFN-γ by these cells occurs between Days 6 and 8 pi [36]. Using tissue culture, it has been demonstrated that the http://parasites.trends.com

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intracellular multiplication of N. caninum is inhibited by IFN-γ [37]. Primary cultured bovine brain cells are highly susceptible to N. caninum infection; however, this growth can be inhibited by the addition of IFN-γ and, to a lesser extent, TNF-α [38]. Cytokines, pregnancy and Neospora caninum

The similarities in cytokine profiles associated with resistance to N. caninum and resistance to other protozoan parasites suggests that the same consequences of infection during pregnancy will hold true. This area to date has not been intensively investigated, but data so far support this contention. Congenital transmission of N. caninum in mice, similar to that of T. gondii, correlates with the day during gestation that the mother is infected [29,39]; tachyzoites are not detected in foetoplacental tissues of BALB/c mice as a result of infection either pre-pregnancy or early in pregnancy [29], but tachyzoites are readily detected in the placenta, in foetal muscle and neural tissue, if mice are infected mid-gestation [29]. Similar results are seen in cattle [40]; cows infected nine weeks before pregnancy do not appear to transmit N. caninum to their calves, whereas cows infected at Week 30 of gestation give birth to asymptomatic, but apparently infected calves (six out of six being seropositive for N. caninum). Foetal loss is also dependent on the time of contraction of infection, relative to insemination. Thus, in BALB/c mice infected 10 days before mating, the ultimate litter size produced is significantly smaller than that for non-infected mice, and in mice infected at Day 5 of gestation, the confirmed foetal resorption rate is almost three times that of non-infected mice (33% versus 12%) [29]. By contrast, no reproductive loss is associated with mice infected with N. caninum in the late stages of pregnancy. Likewise in cattle, infection with N. caninum in early gestation (Week 10) causes foetal loss (five out of six cows lost their foetus at Week 13 of gestation), whereas infection at 30 weeks of gestation has no affect on foetal viability [40]. Cytokines seem likely to play a role in both preventing congenital transmission of N. caninum and inducing abortion, probably in a similar pattern as that proposed for T. gondii [9], although the evidence so far is largely circumstantial. Long and Baszler [41] have investigated the effects of IL-4 on transmission of N. caninum during pregnancy by modulating the level of IL-4 in mice. Pregnant mice were given an injection of IL-4 monoclonal antibody (mAb) to neutralize the effects of maternal IL-4, with an accompanying dose of virulent N. caninum. This did not reduce the rate of vertical transmission. However, mice injected with IL-4 mAb concomitantly with avirulent N. caninum before pregnancy and then challenged during pregnancy with virulent N. caninum, showed a decrease in vertical transmission. This decrease was seen in the numbers of mice transmitting N. caninum to

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offspring and in the frequency of transmission by an individual mouse. The decrease in transmission was associated with lower levels of maternal IL-4 secretion, lower levels of IL-4 mRNA expression and higher levels of IFN-γ secretion. Protected mice showed a decrease in N. caninum-specific IgG1 when compared with non-protected mice. This study demonstrated that modulation of a Th2 cytokine can reduce the frequency of vertical transmission of N. caninum. In cattle, there are currently fewer data on cytokine production in response to N. caninum during pregnancy; infection at various times of gestation results in a biased IgG2 response, References 1 Jankovic, D. et al. (2001) Th1- and Th2-cell commitment during infectious disease: asymmetry in divergent pathways. Trends Immunol. 22, 450–457 2 Hunter, C.A. and Reiner, S.L. (2000) Cytokines and T cells in host defence. Curr. Opin. Immunol. 12, 413–418 3 Wang, Z-H. et al. (1994) CD4+ effector cells default to the Th2 pathway in interferon γ-deficient mice infected with Leishmania major. J. Exp. Med. 179, 1367–1371 4 Raghupathy, R. (1997) Th1-type immunity is incompatible with successful pregnancy. Immunol. Today 18, 478–482 5 Piccini, M.P. et al. (2000) Role of hormone controlled T-cell cytokines in the maintenance of pregnancy. Biochem. Soc. Trans. 28, 212–215 6 Baley, J.E. and Schacter, B.Z. (1985) Mechanisms of diminished natural killer cell activity in pregnant women and neonates. J. Immunol. 134, 3042–3048 7 Miller, L. et al. (1996) Progesterone inhibits inducible nitric oxide synthase gene expression and nitric oxide production in murine macrophages. J. Leukoc. Biol. 59, 442–450 8 Miller, L. and Hunt, J.S. (1998) Regulation of TNF-alpha production in activated mouse macrophages by progesterone. J. Immunol. 160, 5098–5104 9 Roberts, C.W. et al. (2001) Sex-associated hormones and immunity to protozoan parasites. Clin. Microbiol. Rev. 14, 476–488 10 Krishnan, L. et al. (1996) Pregnancy impairs resistance of C57BL/6 mice to Leishmania major infection and causes decreased antigen-specific IFN-γ responses and increased production of T helper 2 cytokines. J. Immunol. 156, 644–652 11 Krishnan, L. et al. (1996) T helper 1 response against Leishmania major in pregnant C56BL/6 mice increases implantation failure and fetal resorptions. J. Immunol. 156, 653–662 12 Louis, J. et al. (1998) Regulation of protective immunity against Leishmania major in mice. Curr. Opin. Immunol. 10, 459–464 13 Scott, P. and Farrell, J.P. (1998) Experimental cutaneous leishmaniasis: induction and regulation of T cells following infection of mice with Leishmania major. Chem. Immunol. 70, 60–80 http://parasites.trends.com

increased lymphoproliferation and enhanced production of IFN-γ by cultured peripheral blood mononuclear cells, all indicative of a Th1 response [40]. Whether this apparent bias influences the outcome of pregnancy remains to be established. However, further study is required because foetal loss and vertical transmission are such significant aspects of infection with N. caninum. Prevention of either of these events may be possible by modulating various components of the immune system, particularly cytokines. Potential vaccine candidates may also be enhanced by a greater understanding of the key components of the immune response during N. caninum infection.

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28 Liddell, S. et al. (1999) Prevention of vertical transfer of Neospora caninum in Balb/c mice by vaccination. J. Parasitol. 85, 1072–1075 29 Long, M.T. and Baszler, T.V. (1996) Fetal loss in BALB/c mice infected with Neospora caninum. J. Parasitol. 82, 608–611 30 Khan, I.A. et al. (1997) Neospora caninum: role for immune cytokines in host immunity. Exp. Parasitol. 85, 24–34 31 Eperon, S. et al. (1999) Susceptibility of B-cell deficient C57BL/6 (µMT) mice to Neospora caninum infection. Parasite Immunol. 21, 225–236 32 Long, M.T. et al. (1998) Comparison of intracerebral parasite load, lesion development, and systemic cytokines in mouse strains infected with Neospora caninum. J. Parasitol. 84, 316–320 33 Baszler, T.V. et al. (1999) Interferon gamma and Interleukin-12 mediate protection to acute Neospora caninum infection in Balb/c mice. Int. J. Parasitol. 29, 1635–1646 34 Tanaka, T. et al. (2000a) The role of CD4+ or CD8+ T cells in the protective immune response of BALB/c mice to Neospora caninum. Vet. Parasitol. 90, 183–191 35 Tanaka, T. et al. (2000b) Growth-inhibitory effects of interferon-gamma on Neospora caninum in murine macrophages by a nitric oxide mechanism. Parasitol. Res. 86, 768–771 36 Lunden, A. et al. (1998) Cellular immune responses in cattle experimentally infected with Neospora caninum. Parasite Immunol. 20, 519–526 37 Innes, E.A. et al. (1995) Interferon gamma inhibits the intracellular multiplication of Neospora caninum, as shown by incorporation of 3H Uracil. J. Comp. Pathol. 113, 95–100 38 Yamane, I. et al. (2000) The inhibitory effect of interferon-gamma and tumor necrosis factor alpha on intracellular multiplication of Neospora caninum in primary bovine brain cells. J. Vet. Med. Sci. 62, 347–351 39 Cole, R.A. et al. (1995) Vertical transmission of Neospora caninum in mice. J. Parasitol. 81, 730–732 40 Williams, D.J.L. et al. (2000) Neospora caninumassociated abortion in cattle: the time of experimentally-induced parasitaemia during gestation determines foetal survival. Parasitology 121, 347–358 41 Long, M.T. and Baszler, T.V. (2000) Neutralisation of maternal IL-4 modulates congenital protozoal transmission: comparison of innate versus acquired immune responses. J. Immunol. 164, 4768–4774