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Immunological control of congenital toxoplasmosis in the murine model
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Immunology Letters 115 (2008) 83–89
Review
Immunological control of congenital toxoplasmosis in the murine model Fiona M. Menzies a , Fiona L. Henriquez b , Craig W. Roberts a,∗ a
Strathclyde Institute of Pharmacy and Biomedical Sciences, 27 Taylor Street, University of Strathclyde, Glasgow G4 0NR, UK b School of Science and Engineering, University of Paisley, Paisley PA1 2BE, UK Received 30 September 2007; received in revised form 9 October 2007; accepted 12 October 2007 Available online 6 November 2007
Abstract Toxoplasmosis is a serious disease in humans where it can cause abortion or congenital infection if a women is exposed to disease for the first time during pregnancy. Infection prior to pregnancy normally results in immunity and which is capable of protecting the foetus. Similar observations have been made in the BALB/c mouse indicating the potential of mice for studying congenital disease. Consequently, the mouse has been used to study how mammals balance the opposing needs of maintaining an immunological environment conducive to successful pregnancy while attempting to control a dangerous pathogen. Moreover the mouse has proven useful for testing the potential of a number of vaccine candidates and adjuvants for their ability to prevent congenital infection and/or reduce foetal death and abortion. © 2007 Elsevier B.V. All rights reserved. Keywords: Toxoplasma gondii; Congenital; Toxoplasmosis; Pregnancy; Vaccine
1. Introduction Toxoplasma gondii is an obligate intracellular protozoan parasite that infects around a third of the human population. T. gondii is normally transmitted by the ingestion of tissue cysts, found in the tissues of infected animals or ingestion of food or water contaminated with oocysts, which are liberated in the faeces of infected cats [1]. Although T. gondii infection may be asymptomatic in immunocompetent individuals it is life-threatening in those immunocompromised due to AIDS or receiving immunosuppressive therapy due to malignancies or post-transplantation [2,3]. When T. gondii is first contracted during pregnancy, congenital transmission can result in infection of the foetus or abortion. Congenital infection may result in foetal abnormalities, including hydrocephalus, intracranial calcifications and retinochoroiditis [2,4,5]. The risk of congenital toxoplasmosis is dependent on a number of factors including the immunological status of the mother, the number and virulence of parasites, and the time of gestation at which infection occurs [6,7]. The risk of congenital transmission is greatest in mothers infected during the third trimester, intermediate in those infected in the second and lowest in those infected in the first. In contrast the risk of abortion
is greatest in those infected during the first trimester and lowest in mothers infected in the third trimester. Disease manifestations are generally most severe in foetuses infected in early gestation. Congenital T. gondii infection occurs not only in humans, but also in livestock where it is more prevalent in pigs, sheep and goats than in poultry or cattle [6]. T. gondii can have significant economic implications for livestock breeders and importers, for example it was estimated that 1.2–2.2% of ewes out of a total of 16 million in the UK abort their foetuses due to T. gondii infection [8]. Current treatment regimes can reduce, but not prevent congenital transmission or abortion [5,9]. Furthermore the relative role of parasite induced damage to the foetus and placenta versus immune mediated abortion is not completely understood. Understanding the role of the immune response in terms of transmission and abortion may be key to designing a safe and effective vaccine capable of preventing congenital transmission and abortion. To facilitate these goals Roberts and Alexander [10] developed a murine model that mimics at least certain aspects of human congenital toxoplasmosis. This review will consider how this particular model has contributed to the understanding of congenital toxoplasmosis and vaccine development. 2. Murine models of congenital toxoplasmosis
∗
Corresponding author. Tel.: +44 141 548 4823. E-mail address: [email protected] (C.W. Roberts).
0165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2007.10.009
Vertical transmission of toxoplasmosis was reported to occur in chronically infected mice and throughout multiple generations
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[11–13]. As healthy humans with chronic infections generally do not transmit T. gondii to their foetuses, this would imply that a fundamental difference between mice and humans would preclude their use for understanding congenital transmission in humans. However, further studies established that in BALB/c mice vertical disease transmission only occurs if the dam is infected for the first time during pregnancy. Furthermore, chronically infected BALB/c mice do not transmit disease even if they are re-exposed to infection during pregnancy [10]. Although this observation was based on studies using the Beverley strain of T. gondii similar results have been confirmed using other type-2 strains such as Prugniaud, Me49 and M3 as well as the type-3 strain and M7741strain [14]. Consequently, the murine model, and in particular the BALB/c mouse has now been extensively used for the purpose of vaccine studies and understanding the interaction of T. gondii infection with the immunological changes that occur during pregnancy. BALB/c mice are in general more resistant to T. gondii infection and habour fewer cysts than susceptible mouse strains such as C57BL/10 [15]. However, this is not the reason for the marked difference between strains in the vertical transmission of T. gondii as chronically infected BALB/K mothers, which harbour numerous cysts, also do not vertically transmit the parasite [10]. 3. The innate immune system controls T. gondii multiplication and directs the development of a protective type-1 response The immune response to T. gondii is complex with many interactions, varies in mouse strains studied, in anatomical sites analysed and is dependent on parasite strain and route of infection (reviewed, [16–20]). However, there are a number of common themes that have emerged from the many models used that are worth summarising before considering how they are influenced by pregnancy. T. gondii possesses a number of immunostimulatory molecules that influence the quality of the innate immune response and consequently the qualities of developing adaptive response. Notably, T. gondii has a number of molecules that contain GPI-anchors, which are effective ligands for TLR2 and 4 [21]. In addition, T. gondii HSP70 has been demonstrated to stimulate macrophages and induce maturation of dendritic cells through ligation of the TLR4 [22,23]. T. gondii profilin interacts with the TLR11 molecule (present only in certain mammalian species including mice, but not humans), to induce IL-12 production. Furthermore, T. gondii cyclophilin 18 has been demonstrated to have the unusual ability to bind the CCR5 receptor and induce IL-12 production [24]. These interactions effectively elicit a robust innate immune response that includes NK cell activation, the production of IFN␥, TNF␣, iNOS expression and consequently reactive nitrogen intermediates that limit parasite multiplication [24]. This innate immune response not only limits initial tachyzoite multiplication, but also effectively drives the development of a type-1 adaptive response including the development of CD8+ cytolytic T cells [25]. CD8+ cytolytic T cells play a major role in resolution of disease and maintenance of immunity in chronically infected mice [26]. The role of type-2
cytokines is multifarious as they antagonise the beneficial effect of the type-1 response in parasite killing, but also play a beneficial role through limiting immune pathology [27]. Thus, IL-4 has been reported to be beneficial and detrimental depending on the model examined [27–31]. IL-10 has been demonstrated to antagonise the ability of macrophages to kill T. gondii, but mice deficient in IL-10 die following T. gondii infection due to cytokine shock [32]. 4. The immune response to T. gondii may favour abortion in pregnant mice Successful pregnancy is associated with a highly regulated immune response in the vicinity of the foetus, a phenomenon that is largely mediated through the production of steroid hormones. Thus although macrophages, NK cells, mast cells, neutrophils and eosinophils are present in the normal deciduas, they do not normally play an adverse role and some may play a role in tissue modelling [33–36]. Mechanisms demonstrated to be responsible for this control of cell function include the ability of steroid hormones to affect their function such as inhibition of nitric oxide, TNF␣, IL-1␣, IL-6 and increase of IL-10 production by macrophages [37–39]. Similarly steroid hormones have been shown to influence mast cell and eosinophil degranulation [40–42]. Numerous studies have demonstrated that NK cell cytotoxicity is reduced during pregnancy [43–46]. Modulation of NK cell activity is critical for successful pregnancy and increased systemic NK activity is associated with spontaneous abortion [47–51]. In addition, NK cell degranulation has been demonstrated to be down-modulated through PIBF which is produced by lymphocytes in response to progesterone. The importance of this molecule is demonstrated by the fact that it can ablate the ability of the progesterone antagonist, RU486 to induce abortion in mice [52]. Furthermore, during pregnancy there is preference towards induction of a type-2 immune response as opposed to a type-1 response. While a type-2 response is compatible with successful pregnancy, a type-1 response may prove disruptive and cause abortion. Again progesterone plays an important role here as it favours the development of Th2 cells and has even been demonstrated to induce Th1 cells to produce IL-4 [53]. Disruption of any of these mechanisms of immunomodulation by T. gondii infection could have detrimental effects for pregnancy as demonstrated in other systems. For example, early studies demonstrated that stimulation of the TLR4 receptor with LPS could cause abortion in mice, which has since been shown to be dependent on TNF␣ and mediated by nitric oxide [49–51]. Similarly, ligation of TLR3 with poly I:C, a synthetic mimic of viral RNA, can induce abortion through a probable NK cell dependent mechanism [54,55]. Notably, a number of T. gondii derived molecules have now been demonstrated to interact with TLRs including HSP70 that is a ligand for TLR4 [23] and likely contribute to iNOS expression and nitric oxide production during T. gondii infection. Furthermore NK cells have been demonstrated to play important roles in protection against adult acquired T. gondii infection and
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their activation could also have adverse effects on pregnancy [49]. Infections such as malaria and Neospora caninum which induce a strong Th1 response have been noted to induce abortion and increase implantation failure [56,57]. For example, in C57BL6 mice, Leishmania major infection induces a robust Th1 response that ablates the pregnancy induced Th2 bias and increases the rate of embryo implantation failure [58]. T. gondii infection induces a strong T cell mediated immune response, with IL-12 release from infected host cells inducing a bias towards a Th1-type response. Dendritic cells are potent inducers of IL-12 production [59] and recent studies have found that in the murine system the T. gondii proteins cyclophilin-18 and profilin bind to the receptors CCR5 and TLR11, respectively, on DCs, thus promoting the production of IL-12 [24,60,61]. This results in a robust Th1 response with IFN␥ production. A recent study found that IFN␥ deficient mice had reduced abortion in spite of having increased numbers of parasites in their uterus and placentas of their foetuses compared with WT mice [62]. This indicates that T. gondii induced IFN␥ can have adverse effects on pregnancy. 5. Pregnancy mediated immune modulation may favour T. gondii multiplication and congenital transmission The ability of pregnancy to modulate the immune response may interfere with the mechanisms that normally control parasite multiplication and assist in congenital infection. However, although the ability of progesterone to reduce iNOS expression and nitric oxide production might be predicted to prevent parasite killing this does not seem to be important at least in vivo models [63]. In contrast, NK cell modulation may be important as depletion of NK cells increases congenital transmission in RAG2−/− mice [64]. However, in BALB/c mice, T and/or B cells might play compensatory roles as similar depletion of NK cells had no effect on congenital transmission [65]. Importantly, NK cells isolated from pregnant mice infected with T. gondii have reduced cytotoxic ability [66]. This imbalance in the production of Th1 and Th2-type cytokines is considered to contribute to the risk of T. gondii transmission to the foetus. The Th2 type cytokines IL-4, IL-5 and IL-10 are constitutively secreted during all three trimesters of pregnancy by cells of the foetal-placental unit, whereas IFN␥ is only detected in the first and second trimesters of pregnancy [67]. This has implications with regard to the susceptibility of mice to T. gondii infection and transplacental transmission of the parasite. Notably, pregnant mice are more susceptible to infection with T. gondii demonstrating increased mortality over nonpregnant female mice a trait that correlates with reduced IFN␥ production [68,69]. Conversely, survival of pregnant mice can be increased by administration of either IFN␥ or IL-2 [69,70]. Furthermore, studies in BALB/c mice demonstrated, through the use of anti-IFN␥ antibodies, that this Th1-type cytokine is important for preventing congenital toxoplasmosis during re-infection [65]. CD8+ and not CD4+ T cells would appear to be critical as their depletion resulted in decreased IFN␥, increased maternal parasiteamia and enhanced congenital transmission [65]. IFN␥
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deficient BALB/c and C57BL6 mice have increased parasites in their uteri and placentas compared with WT mice [62]. In contrast the effect of IL-4 would appear to be mouse strain dependent as BALB/c mice deficient in IL-4 have reduced congenital transmission compared with WT mice of the same strain, whereas no difference in transmission is observed in B6/129 mice [31,71]. 6. Correlations between mice and humans? Although exceptions have been reported (for example, [72]), generally in humans infection with T. gondii prior to pregnancy will confer protection to the foetus upon re-infection [6]. However, should infection occur for the first time during pregnancy, there is a risk of foetal disease transmission. The trimester of pregnancy at which infection occurs for the first time will determine the outcome of congenital infection [6]. For example, it is generally considered that if a woman is infected during the first trimester when immune modulation is less pronounced, there is a relatively high risk of abortion. In contrast, infection during the third trimester poses a relatively low risk of abortion, but high risk of congenital transmission [4,7]. Thus, the actual risk of T. gondii transmission from mother to the foetus is inversely related to the abortion rate, with there being a higher risk of the parasite passing to the foetus in the later stages of the pregnancy than at the beginning. Surviving foetuses born to mothers infected during their first trimester have a high risk of severe congenital toxoplasmosis, whereas those born to mothers infected in the third trimester often have mild or sub-clinical infection, which may develop into clinical symptoms later in life [2,6]. As in humans, chronically infected BALB/c mice do not transmit disease to their foetuses even if re-exposed to infection. Moreover, infection of BALB/c mice in the first trimester, as observed in humans, results in embryo loss due to abortion or resorption, whereas infection in the second trimester results in congenital transmission. Again similar to the situation observed in humans, infection in the third trimester has been observed to induce abortion. However, as pregnancy in the mouse is only 21 days, infection in the third trimester would not appear to give sufficient time for congenital transmission to occur. 7. Vaccine development and testing of antimicrobial agents The murine model of congenital toxoplasmosis, predominantly the BALB/c mouse, has been used to test the efficacy of potential vaccines and antimicrobials (Table 1). However, vaccination studies have also been carried out in other mouse strains, Swiss Webster, C57BL/6 and OF1 Swiss mice [73–75]. The ability of these mouse strains to develop solid immunity capable of preventing vertical disease transmission similar to humans and BALB/c mouse remains to be determined. In addition, prior to the BALB/c model, vaccination studies indicated that a degree of immunity could be induced in a mouse model of congenital infection [73]. In these pioneering vaccination studies, Swiss Webster mice were successfully immunised either subcutaneously or intraintestinally with the ts (temperature sensitive) -4 strain of T.
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Table 1 Summary of vaccination studies using the murine model of congenital toxoplasmosis Mouse strain
T. gondii strain (route of infection)
Vaccine (route of inoculation)
Effect on congenital transmission
Effect on abortion
Effect on immune response
Reference
Swiss
Me49 (oral)
ts4 (subcutaneous)
No effect
Not reported
[73]
Swiss
Me49 (oral)
ts4 (intraintestinal)
Not reported
BALB/c
Beverley (oral)
BALB/c
Beverley (oral)
STAg/NISV (sub-cutaneous) STAg/FCA (sub-cutaneous)
Reduced (not significant) Reduced Reduced
Reduced
BALB/c
Beverley (oral)
STAg (sub-cutaneous)
Reduced
Reduced
BALB/c
P (oral) and C4 (oral)
Reduced
Reduced
BALB/c
Beverley (oral)
No effect
No effect
BALB/c
Me49 (oral)
L/STAg, L/SCAg L/STCAg, L/pTAg STAg, FCA/STAg SAG1 gene in plasmid (intra-muscular) SAG1 gene in plasmid (intra-muscular)
Reduced
Not reported
CBA
Me49 (oral)
SAG1 gene in plasmid (intra-muscular)
No effect
Not reported
OF1
76K (oral)
Reduced
Reduced
OF1
76K (orally)
GRA4 and SAG1 associated with GM-CSF plasmid T. gondii (RH strain) deficient in Mic1-3
Reduced
Reduced
Antibody production; no enhanced macrophage activity No antibody; no enhanced macrophage activity Increased ex vivo splenocyte production of IFN␥ compared with non-vaccinated mice No significant difference in IFN␥ production compared with non-vaccinated mice No significant difference in IFN␥ production compared with non-vaccinated mice Increased splenocyte proliferation and antibody production compared with non-vaccinated mice Increased ex vivo splenocyte production of IFN␥ compared with non-vaccinated mice Increased serum IFN␥ and IL-10 production compared with non-vaccinated controls. Similar levels of IL-4 in vaccinated and non-vaccinated mice Increased IL-4 and IL-10 production in vaccinated animals compared with non-vaccinated Similar levels of IFN␥ in vaccinated and non-vaccinated mice Increased serum IFN␥ and IL-10 production compared with non-vaccinated controls Increased IFN␥, IL-2, IL-10
gondii against either M7741 tachyzoites or Me49 bradyzoites [73]. Subcutaneous inoculation with either soluble T. gondii tachyzoite antigen (STAg) emulsified with Freund’s complete adjuvant or STAg in non-ionic surfactant vesicles (NISV) was shown to be effective in inducing protection in pregnant dams, such that pups born to vaccinated mothers survived to maturity [76]. This protection was associated with enhancement of T cell produced IFN␥ following stimulation in vitro with STAg. A further study examined the efficacy of STAg, purified tachyzoite antigen, cyst antigens and a cocktail of antigens from different life cycle stages in various combinations with different adjuvants. Similar to reported by Roberts et al. [76] liposomally entrapped antigen reduced foetal death and the incidence of congenital transmission, whereas immunisation with antigen in PBS or emulsified in FCA had no beneficial effect [77]. Thus the adjuvant system used in the immunisation process would appear to be of paramount importance with vesicular systems having best results to date. Protection against congenital toxoplasmosis in BALB/c mice has also been demonstrated with vaccination with single recombinant protein. Letscher-Bru et al. [78] reported that vaccination with the surface protein SAG1 reduced foetal infection in BALB/c mice. This was associated with increased IFN␥ in the maternal serum of vaccinated mice compared with control mice on day 19 of pregnancy. Interestingly, the efficacy of this vaccine
Reduced
[76]
[77]
[79] [78]
[74]
[75]
would appear to be mouse strain dependent as in the same study no protection or increase in serum IFN␥ was observed in similarly treated CBA mice. The method of vaccination again would appear important as a further study that used DNA vaccination with the SAG1 gene in BALB/c mice found that no protection was afforded against adult acquired T. gondii infection, but not maternofoetal transmission [79]. DNA vaccination with other T. gondii derived genes has provided some protection against foetal infection. Thus reduced foetal death was observed in outbred OF1 mice immunised with a cocktail of plasmids encoding the T. gondii derived antigens GRA4 and SAG1 in the presence of a plasmid encoding murine GM-CSF when challenged orally with 76K strain of T. gondii [74]. As this was the only combination of plasmids tested in this study, the role of he individual components in preventing foetal death remains to be determined. The ability to genetically engineer T. gondii auxotrophs or variants that have reduced virulence has provided the opportunity to develop live attenuated vaccines. A genetically modified T. gondii (RH strain), deficient in two microneme genes which normally function to assist in the adhesion and invasion of host cells has been generated. These parasites, Mic1-3-KO, have been shown to reduce the incidence and severity of congenital transmission of OF1 mice challenged with the 76K strain of T. gondii [75]. Immunity was associated with enhanced maternal Th1 responses, specifically increased
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IFN␥ production by tachyzoite antigen stimulated splenocytes from those mice which had been previously vaccinated with Mic1-3-KO compared with non-vaccinated control mice. However, IL-10 levels were also increased in the vaccinated mice, which might be significant in preventing abortion or foetal death. 8. Conclusions The murine model has proven useful for understanding congenital toxoplasmosis, but also some limitations for studying congenital toxoplasmosis in humans. In particular the BALB/c mouse has proven particularly useful as this strain of mouse can develop immunity capable of preventing congenital disease transmission as observed in humans. The murine model has been employed to dissect the role of parasite multiplication and the immune response on foetal death and disease transmission for vaccination. It has also been effective to test the ability of chemotherapeutics to limit vertical disease transmission [80]. The relatively short gestation time facilitates reasonably efficient experimentation, but is also a limitation. Specifically, it is challenging to study the effect of immune modulation in the third trimester of pregnancy on parasite multiplication or vice versa. However, the availability of numerous immunological reagents and the continued expansion of the catalogue of gene deficient mice will ensure its continued utility and should provide increased understanding of the pathogenesis of congenital T. gondii infection. References [1] Dubey JP. Sources of Toxoplasma gondii infection in pregnancy. BMJ 2000;321:127–212. [2] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004;363:1965–76. [3] Sensini A. Toxoplasma gondii infection in pregnancy: opportunities and pitfalls of serological diagnosis. Clin Microbiol Infect 2006;12:504–12. [4] Dunn D, Wallon M, Peyron F, Peterson E, Peckham C, Gilbert R. Mother-tochild transmission of toxoplasmosis: risk estimates for clinical counselling. Lancet 1999;353:1829–33. [5] Wallon M, Liou C, Garner P, Peyron F. Congenital toxoplasmosis: systematic review of efficacy of treatment in pregnancy. BMJ 1999;318:1511–4. [6] Tenter AM, Heckeroth AJ, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2000;30:1217–58. [7] Pelloux H, Brenier-Pinchart MP, Fricker-Hidalgo H. Protozoan infections in humans: congenital toxoplasmosis. Eur J Protistol 2003;39:444–8. [8] Bennett R, Christiansen K, Clifton-Hadley R. Preliminary estimates of the direct costs associated with endemic diseases of livestock in Great Britain. Prev Vet Med 1999;39:155–71. [9] Guerina NG, Hsu HW, Meissner C, Maguire JH, Lynfield R, Stechenberg B, et al. Neonatal serologic screening and early treatment for congenital Toxoplasma gondii infection. N Engl J Med 1994;330:1858–63. [10] Roberts CW, Alexander J. Studies on a murine model of congenital toxoplasmosis: vertical disease transmission only occurs in BALB/c mice infected for the first time during pregnancy. Parasitology 1992;104:19–23. [11] Beverley JK. Congenital transmission of toxoplasmosis through successive generations of mice. Nature 1959;183:1348–9. [12] Remington JS. Experiments on the transmission of toxoplasmosis. Surv Ophthalmol 1961;6:856–76. [13] De Roever-Bonnet H. Congenital toxoplasma infections in mice and hamsters infected with avirulent and virulent strains. Trop Geogr Med 1969;21:443–50.
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[37] Miller L, Alley EW, Murphy WJ, Russell SW, Hunt JS. Progesterone inhibits inducible nitric oxide synthase gene expression and nitric oxide production in murine macrophages. J Leukoc Biol 1996;59:442–50. [38] Miller L, Hunt JS. Sex steroid hormones and macrophage function. Life Sci 1996;59:1–14. [39] Chao TC, Chao HH, Chen MF, Greager JA, Walter RJ. Female sex hormones modulate the function of LPS-treated macrophages. Am J Reprod Immunol 2000;44:310–8. [40] Hamano N, Terada N, Maesako K, Numata T, Konno A. Effect of sex hormones on eosinophilic inflammation in nasal mucosa. Allergy Asthma Proc 1998;19:263–9. [41] Silva H, Tchernitchin AN, Tchernitchin NN. Low doses of estradiol-17 alpha degranulate blood eosinophil leucocytes and high doses alter homeostatic mechanisms. Med Sci Res 1997;25:201–4. [42] Vliagoftis H, Dimitriadou V, Boucher W, Rozniecki JJ, Correia I, Raam S, et al. Estradiol augments while tamoxifen inhibits rat mast-cell secretion. Int Arch Allergy Immunol 1992;98:398–409. [43] Baley JE, Schacter BZ. Mechanisms of diminished natural killer cell activity in pregnant women and neonates. J Immunol 1985;134:3042–8. [44] Furukawa K, Itoh K, Okamura K, Kumagai K, Suzuki M. Changes in NK cell activity during the oestrous cycle and pregnancy in mice. J Reprod Immunol 1984;6:353–63. [45] Toder V, Nebel L, Elrad H, Blank M, Durdana A, Gleicher N. Studies of natural killer cells in pregnancy. II. The immunoregulatory effect of pregnancy substances. J Clin Lab Immunol 1984;14:129–33. [46] Toder V, Nebel L, Gleicher N. Studies of natural killer cells in pregnancy. I. Analysis at the single cell level. J Clin Lab Immunol 1984;14:123–7. [47] Coulam CB, Goodman C, Roussev RG, Thomason EJ, Beaman KD. Systemic CD561 cells can predict pregnancy outcome. Am J Reprod Immunol 1995;33:40–6. [48] Szekeres-Bartho J, Varga P, Pacsa AS. Immunologic factors contributing to the initiation of labor-lymphocyte reactivity in term labor and threatened preterm delivery. Am J Obstet Gynecol 1986;155:108–12. [49] Gendron RL, Baines MG. Infiltrating decidual natural killer cells are associated with spontaneous abortion in mice. Cell Immunol 1988;113: 261–7. [50] Gendron RL, Farookhi R, Baines MG. Resorption of CBA/J3 DBA/2 mouse conceptuses in CBA/J uteri correlates with failure if the feto-placental unit to suppress natural killer cell activity. J Reprod Fertil 1990;89:277–84. [51] Gendron RL. Lipopolysaccharide-induced fetal resorption in mice is associated with intrauterine production of tumour necrosis factor-alpha. J Reprod Fertil 1990;90:395–402. [52] Szekeres-Bartho J, Par G, Dombay G, Smart YC, Volgyi Z. The antiabortive effect of progesterone-induced blocking factor in mice is manifested by modulating NK activity. Cell Immunol 1997;177:194–9. [53] Piccinni MP, Giudizi MG, Biagiotti R, Beloni L, Giannarini L, Sampognaro S, et al. Progesterone favors the development of human T-helper cells producing Th2-type cytokines and promotes both IL-4 production and membrane cd30 expression in established Th1 cell clones. J Immunol 1995;155:128–33. [54] Lin Y, Zeng Y, Zeng S, Wang T. Potential role of toll-like receptor 3 in a murine model of polyinosinic-polycytidylic acid-induced embryo resorption. Fertil Steril Suppl 2006;1:1125–9. [55] Zhang J, Wei H, Wu D, Tian Z. Toll-like receptor 3 agonist induces impairment of uterine vascular remodeling and fetal losses in CBA x DBA/2 mice. J Reprod Immunol 2007;74:61–7. [56] Fried M, Muga RO. Misore AO Duffy PE. Malaria elicits type 1 cytokines in the human placenta: IFN-gamma and TNF-alpha associated with pregnancy outcomes. J Immunol 1998;160:2523–30. [57] Quinn HE, Ellis JT, Smith NC. Neospora caninum: a cause of immunemediated failure of pregnancy? Trends Parasitol 2003;18:391–4. [58] Krishnan L, Guilbert LJ, Wegmann TG, Belosevic M, Mosmann TR. T helper 1 response against Leishmania major in pregnant C57BL/6 mice increases implantation failure and fetal resorptions. Correlation with increased IFN-gamma and TNF and reduced IL-10 production by placental cells. J Immunol 1996;156:653–62. [59] Koch F, Stanzl U, Jennewein P, Janke K, Heufler C, Kampgen E, et al. High level IL-12 production by murine dendritic cells: upregulation via MHC
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Immunology Letters 115 (2008) 83–89
Review
Immunological control of congenital toxoplasmosis in the murine model Fiona M. Menzies a , Fiona L. Henriquez b , Craig W. Roberts a,∗ a
Strathclyde Institute of Pharmacy and Biomedical Sciences, 27 Taylor Street, University of Strathclyde, Glasgow G4 0NR, UK b School of Science and Engineering, University of Paisley, Paisley PA1 2BE, UK Received 30 September 2007; received in revised form 9 October 2007; accepted 12 October 2007 Available online 6 November 2007
Abstract Toxoplasmosis is a serious disease in humans where it can cause abortion or congenital infection if a women is exposed to disease for the first time during pregnancy. Infection prior to pregnancy normally results in immunity and which is capable of protecting the foetus. Similar observations have been made in the BALB/c mouse indicating the potential of mice for studying congenital disease. Consequently, the mouse has been used to study how mammals balance the opposing needs of maintaining an immunological environment conducive to successful pregnancy while attempting to control a dangerous pathogen. Moreover the mouse has proven useful for testing the potential of a number of vaccine candidates and adjuvants for their ability to prevent congenital infection and/or reduce foetal death and abortion. © 2007 Elsevier B.V. All rights reserved. Keywords: Toxoplasma gondii; Congenital; Toxoplasmosis; Pregnancy; Vaccine
1. Introduction Toxoplasma gondii is an obligate intracellular protozoan parasite that infects around a third of the human population. T. gondii is normally transmitted by the ingestion of tissue cysts, found in the tissues of infected animals or ingestion of food or water contaminated with oocysts, which are liberated in the faeces of infected cats [1]. Although T. gondii infection may be asymptomatic in immunocompetent individuals it is life-threatening in those immunocompromised due to AIDS or receiving immunosuppressive therapy due to malignancies or post-transplantation [2,3]. When T. gondii is first contracted during pregnancy, congenital transmission can result in infection of the foetus or abortion. Congenital infection may result in foetal abnormalities, including hydrocephalus, intracranial calcifications and retinochoroiditis [2,4,5]. The risk of congenital toxoplasmosis is dependent on a number of factors including the immunological status of the mother, the number and virulence of parasites, and the time of gestation at which infection occurs [6,7]. The risk of congenital transmission is greatest in mothers infected during the third trimester, intermediate in those infected in the second and lowest in those infected in the first. In contrast the risk of abortion
is greatest in those infected during the first trimester and lowest in mothers infected in the third trimester. Disease manifestations are generally most severe in foetuses infected in early gestation. Congenital T. gondii infection occurs not only in humans, but also in livestock where it is more prevalent in pigs, sheep and goats than in poultry or cattle [6]. T. gondii can have significant economic implications for livestock breeders and importers, for example it was estimated that 1.2–2.2% of ewes out of a total of 16 million in the UK abort their foetuses due to T. gondii infection [8]. Current treatment regimes can reduce, but not prevent congenital transmission or abortion [5,9]. Furthermore the relative role of parasite induced damage to the foetus and placenta versus immune mediated abortion is not completely understood. Understanding the role of the immune response in terms of transmission and abortion may be key to designing a safe and effective vaccine capable of preventing congenital transmission and abortion. To facilitate these goals Roberts and Alexander [10] developed a murine model that mimics at least certain aspects of human congenital toxoplasmosis. This review will consider how this particular model has contributed to the understanding of congenital toxoplasmosis and vaccine development. 2. Murine models of congenital toxoplasmosis
∗
Corresponding author. Tel.: +44 141 548 4823. E-mail address: [email protected] (C.W. Roberts).
0165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2007.10.009
Vertical transmission of toxoplasmosis was reported to occur in chronically infected mice and throughout multiple generations
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[11–13]. As healthy humans with chronic infections generally do not transmit T. gondii to their foetuses, this would imply that a fundamental difference between mice and humans would preclude their use for understanding congenital transmission in humans. However, further studies established that in BALB/c mice vertical disease transmission only occurs if the dam is infected for the first time during pregnancy. Furthermore, chronically infected BALB/c mice do not transmit disease even if they are re-exposed to infection during pregnancy [10]. Although this observation was based on studies using the Beverley strain of T. gondii similar results have been confirmed using other type-2 strains such as Prugniaud, Me49 and M3 as well as the type-3 strain and M7741strain [14]. Consequently, the murine model, and in particular the BALB/c mouse has now been extensively used for the purpose of vaccine studies and understanding the interaction of T. gondii infection with the immunological changes that occur during pregnancy. BALB/c mice are in general more resistant to T. gondii infection and habour fewer cysts than susceptible mouse strains such as C57BL/10 [15]. However, this is not the reason for the marked difference between strains in the vertical transmission of T. gondii as chronically infected BALB/K mothers, which harbour numerous cysts, also do not vertically transmit the parasite [10]. 3. The innate immune system controls T. gondii multiplication and directs the development of a protective type-1 response The immune response to T. gondii is complex with many interactions, varies in mouse strains studied, in anatomical sites analysed and is dependent on parasite strain and route of infection (reviewed, [16–20]). However, there are a number of common themes that have emerged from the many models used that are worth summarising before considering how they are influenced by pregnancy. T. gondii possesses a number of immunostimulatory molecules that influence the quality of the innate immune response and consequently the qualities of developing adaptive response. Notably, T. gondii has a number of molecules that contain GPI-anchors, which are effective ligands for TLR2 and 4 [21]. In addition, T. gondii HSP70 has been demonstrated to stimulate macrophages and induce maturation of dendritic cells through ligation of the TLR4 [22,23]. T. gondii profilin interacts with the TLR11 molecule (present only in certain mammalian species including mice, but not humans), to induce IL-12 production. Furthermore, T. gondii cyclophilin 18 has been demonstrated to have the unusual ability to bind the CCR5 receptor and induce IL-12 production [24]. These interactions effectively elicit a robust innate immune response that includes NK cell activation, the production of IFN␥, TNF␣, iNOS expression and consequently reactive nitrogen intermediates that limit parasite multiplication [24]. This innate immune response not only limits initial tachyzoite multiplication, but also effectively drives the development of a type-1 adaptive response including the development of CD8+ cytolytic T cells [25]. CD8+ cytolytic T cells play a major role in resolution of disease and maintenance of immunity in chronically infected mice [26]. The role of type-2
cytokines is multifarious as they antagonise the beneficial effect of the type-1 response in parasite killing, but also play a beneficial role through limiting immune pathology [27]. Thus, IL-4 has been reported to be beneficial and detrimental depending on the model examined [27–31]. IL-10 has been demonstrated to antagonise the ability of macrophages to kill T. gondii, but mice deficient in IL-10 die following T. gondii infection due to cytokine shock [32]. 4. The immune response to T. gondii may favour abortion in pregnant mice Successful pregnancy is associated with a highly regulated immune response in the vicinity of the foetus, a phenomenon that is largely mediated through the production of steroid hormones. Thus although macrophages, NK cells, mast cells, neutrophils and eosinophils are present in the normal deciduas, they do not normally play an adverse role and some may play a role in tissue modelling [33–36]. Mechanisms demonstrated to be responsible for this control of cell function include the ability of steroid hormones to affect their function such as inhibition of nitric oxide, TNF␣, IL-1␣, IL-6 and increase of IL-10 production by macrophages [37–39]. Similarly steroid hormones have been shown to influence mast cell and eosinophil degranulation [40–42]. Numerous studies have demonstrated that NK cell cytotoxicity is reduced during pregnancy [43–46]. Modulation of NK cell activity is critical for successful pregnancy and increased systemic NK activity is associated with spontaneous abortion [47–51]. In addition, NK cell degranulation has been demonstrated to be down-modulated through PIBF which is produced by lymphocytes in response to progesterone. The importance of this molecule is demonstrated by the fact that it can ablate the ability of the progesterone antagonist, RU486 to induce abortion in mice [52]. Furthermore, during pregnancy there is preference towards induction of a type-2 immune response as opposed to a type-1 response. While a type-2 response is compatible with successful pregnancy, a type-1 response may prove disruptive and cause abortion. Again progesterone plays an important role here as it favours the development of Th2 cells and has even been demonstrated to induce Th1 cells to produce IL-4 [53]. Disruption of any of these mechanisms of immunomodulation by T. gondii infection could have detrimental effects for pregnancy as demonstrated in other systems. For example, early studies demonstrated that stimulation of the TLR4 receptor with LPS could cause abortion in mice, which has since been shown to be dependent on TNF␣ and mediated by nitric oxide [49–51]. Similarly, ligation of TLR3 with poly I:C, a synthetic mimic of viral RNA, can induce abortion through a probable NK cell dependent mechanism [54,55]. Notably, a number of T. gondii derived molecules have now been demonstrated to interact with TLRs including HSP70 that is a ligand for TLR4 [23] and likely contribute to iNOS expression and nitric oxide production during T. gondii infection. Furthermore NK cells have been demonstrated to play important roles in protection against adult acquired T. gondii infection and
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their activation could also have adverse effects on pregnancy [49]. Infections such as malaria and Neospora caninum which induce a strong Th1 response have been noted to induce abortion and increase implantation failure [56,57]. For example, in C57BL6 mice, Leishmania major infection induces a robust Th1 response that ablates the pregnancy induced Th2 bias and increases the rate of embryo implantation failure [58]. T. gondii infection induces a strong T cell mediated immune response, with IL-12 release from infected host cells inducing a bias towards a Th1-type response. Dendritic cells are potent inducers of IL-12 production [59] and recent studies have found that in the murine system the T. gondii proteins cyclophilin-18 and profilin bind to the receptors CCR5 and TLR11, respectively, on DCs, thus promoting the production of IL-12 [24,60,61]. This results in a robust Th1 response with IFN␥ production. A recent study found that IFN␥ deficient mice had reduced abortion in spite of having increased numbers of parasites in their uterus and placentas of their foetuses compared with WT mice [62]. This indicates that T. gondii induced IFN␥ can have adverse effects on pregnancy. 5. Pregnancy mediated immune modulation may favour T. gondii multiplication and congenital transmission The ability of pregnancy to modulate the immune response may interfere with the mechanisms that normally control parasite multiplication and assist in congenital infection. However, although the ability of progesterone to reduce iNOS expression and nitric oxide production might be predicted to prevent parasite killing this does not seem to be important at least in vivo models [63]. In contrast, NK cell modulation may be important as depletion of NK cells increases congenital transmission in RAG2−/− mice [64]. However, in BALB/c mice, T and/or B cells might play compensatory roles as similar depletion of NK cells had no effect on congenital transmission [65]. Importantly, NK cells isolated from pregnant mice infected with T. gondii have reduced cytotoxic ability [66]. This imbalance in the production of Th1 and Th2-type cytokines is considered to contribute to the risk of T. gondii transmission to the foetus. The Th2 type cytokines IL-4, IL-5 and IL-10 are constitutively secreted during all three trimesters of pregnancy by cells of the foetal-placental unit, whereas IFN␥ is only detected in the first and second trimesters of pregnancy [67]. This has implications with regard to the susceptibility of mice to T. gondii infection and transplacental transmission of the parasite. Notably, pregnant mice are more susceptible to infection with T. gondii demonstrating increased mortality over nonpregnant female mice a trait that correlates with reduced IFN␥ production [68,69]. Conversely, survival of pregnant mice can be increased by administration of either IFN␥ or IL-2 [69,70]. Furthermore, studies in BALB/c mice demonstrated, through the use of anti-IFN␥ antibodies, that this Th1-type cytokine is important for preventing congenital toxoplasmosis during re-infection [65]. CD8+ and not CD4+ T cells would appear to be critical as their depletion resulted in decreased IFN␥, increased maternal parasiteamia and enhanced congenital transmission [65]. IFN␥
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deficient BALB/c and C57BL6 mice have increased parasites in their uteri and placentas compared with WT mice [62]. In contrast the effect of IL-4 would appear to be mouse strain dependent as BALB/c mice deficient in IL-4 have reduced congenital transmission compared with WT mice of the same strain, whereas no difference in transmission is observed in B6/129 mice [31,71]. 6. Correlations between mice and humans? Although exceptions have been reported (for example, [72]), generally in humans infection with T. gondii prior to pregnancy will confer protection to the foetus upon re-infection [6]. However, should infection occur for the first time during pregnancy, there is a risk of foetal disease transmission. The trimester of pregnancy at which infection occurs for the first time will determine the outcome of congenital infection [6]. For example, it is generally considered that if a woman is infected during the first trimester when immune modulation is less pronounced, there is a relatively high risk of abortion. In contrast, infection during the third trimester poses a relatively low risk of abortion, but high risk of congenital transmission [4,7]. Thus, the actual risk of T. gondii transmission from mother to the foetus is inversely related to the abortion rate, with there being a higher risk of the parasite passing to the foetus in the later stages of the pregnancy than at the beginning. Surviving foetuses born to mothers infected during their first trimester have a high risk of severe congenital toxoplasmosis, whereas those born to mothers infected in the third trimester often have mild or sub-clinical infection, which may develop into clinical symptoms later in life [2,6]. As in humans, chronically infected BALB/c mice do not transmit disease to their foetuses even if re-exposed to infection. Moreover, infection of BALB/c mice in the first trimester, as observed in humans, results in embryo loss due to abortion or resorption, whereas infection in the second trimester results in congenital transmission. Again similar to the situation observed in humans, infection in the third trimester has been observed to induce abortion. However, as pregnancy in the mouse is only 21 days, infection in the third trimester would not appear to give sufficient time for congenital transmission to occur. 7. Vaccine development and testing of antimicrobial agents The murine model of congenital toxoplasmosis, predominantly the BALB/c mouse, has been used to test the efficacy of potential vaccines and antimicrobials (Table 1). However, vaccination studies have also been carried out in other mouse strains, Swiss Webster, C57BL/6 and OF1 Swiss mice [73–75]. The ability of these mouse strains to develop solid immunity capable of preventing vertical disease transmission similar to humans and BALB/c mouse remains to be determined. In addition, prior to the BALB/c model, vaccination studies indicated that a degree of immunity could be induced in a mouse model of congenital infection [73]. In these pioneering vaccination studies, Swiss Webster mice were successfully immunised either subcutaneously or intraintestinally with the ts (temperature sensitive) -4 strain of T.
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Table 1 Summary of vaccination studies using the murine model of congenital toxoplasmosis Mouse strain
T. gondii strain (route of infection)
Vaccine (route of inoculation)
Effect on congenital transmission
Effect on abortion
Effect on immune response
Reference
Swiss
Me49 (oral)
ts4 (subcutaneous)
No effect
Not reported
[73]
Swiss
Me49 (oral)
ts4 (intraintestinal)
Not reported
BALB/c
Beverley (oral)
BALB/c
Beverley (oral)
STAg/NISV (sub-cutaneous) STAg/FCA (sub-cutaneous)
Reduced (not significant) Reduced Reduced
Reduced
BALB/c
Beverley (oral)
STAg (sub-cutaneous)
Reduced
Reduced
BALB/c
P (oral) and C4 (oral)
Reduced
Reduced
BALB/c
Beverley (oral)
No effect
No effect
BALB/c
Me49 (oral)
L/STAg, L/SCAg L/STCAg, L/pTAg STAg, FCA/STAg SAG1 gene in plasmid (intra-muscular) SAG1 gene in plasmid (intra-muscular)
Reduced
Not reported
CBA
Me49 (oral)
SAG1 gene in plasmid (intra-muscular)
No effect
Not reported
OF1
76K (oral)
Reduced
Reduced
OF1
76K (orally)
GRA4 and SAG1 associated with GM-CSF plasmid T. gondii (RH strain) deficient in Mic1-3
Reduced
Reduced
Antibody production; no enhanced macrophage activity No antibody; no enhanced macrophage activity Increased ex vivo splenocyte production of IFN␥ compared with non-vaccinated mice No significant difference in IFN␥ production compared with non-vaccinated mice No significant difference in IFN␥ production compared with non-vaccinated mice Increased splenocyte proliferation and antibody production compared with non-vaccinated mice Increased ex vivo splenocyte production of IFN␥ compared with non-vaccinated mice Increased serum IFN␥ and IL-10 production compared with non-vaccinated controls. Similar levels of IL-4 in vaccinated and non-vaccinated mice Increased IL-4 and IL-10 production in vaccinated animals compared with non-vaccinated Similar levels of IFN␥ in vaccinated and non-vaccinated mice Increased serum IFN␥ and IL-10 production compared with non-vaccinated controls Increased IFN␥, IL-2, IL-10
gondii against either M7741 tachyzoites or Me49 bradyzoites [73]. Subcutaneous inoculation with either soluble T. gondii tachyzoite antigen (STAg) emulsified with Freund’s complete adjuvant or STAg in non-ionic surfactant vesicles (NISV) was shown to be effective in inducing protection in pregnant dams, such that pups born to vaccinated mothers survived to maturity [76]. This protection was associated with enhancement of T cell produced IFN␥ following stimulation in vitro with STAg. A further study examined the efficacy of STAg, purified tachyzoite antigen, cyst antigens and a cocktail of antigens from different life cycle stages in various combinations with different adjuvants. Similar to reported by Roberts et al. [76] liposomally entrapped antigen reduced foetal death and the incidence of congenital transmission, whereas immunisation with antigen in PBS or emulsified in FCA had no beneficial effect [77]. Thus the adjuvant system used in the immunisation process would appear to be of paramount importance with vesicular systems having best results to date. Protection against congenital toxoplasmosis in BALB/c mice has also been demonstrated with vaccination with single recombinant protein. Letscher-Bru et al. [78] reported that vaccination with the surface protein SAG1 reduced foetal infection in BALB/c mice. This was associated with increased IFN␥ in the maternal serum of vaccinated mice compared with control mice on day 19 of pregnancy. Interestingly, the efficacy of this vaccine
Reduced
[76]
[77]
[79] [78]
[74]
[75]
would appear to be mouse strain dependent as in the same study no protection or increase in serum IFN␥ was observed in similarly treated CBA mice. The method of vaccination again would appear important as a further study that used DNA vaccination with the SAG1 gene in BALB/c mice found that no protection was afforded against adult acquired T. gondii infection, but not maternofoetal transmission [79]. DNA vaccination with other T. gondii derived genes has provided some protection against foetal infection. Thus reduced foetal death was observed in outbred OF1 mice immunised with a cocktail of plasmids encoding the T. gondii derived antigens GRA4 and SAG1 in the presence of a plasmid encoding murine GM-CSF when challenged orally with 76K strain of T. gondii [74]. As this was the only combination of plasmids tested in this study, the role of he individual components in preventing foetal death remains to be determined. The ability to genetically engineer T. gondii auxotrophs or variants that have reduced virulence has provided the opportunity to develop live attenuated vaccines. A genetically modified T. gondii (RH strain), deficient in two microneme genes which normally function to assist in the adhesion and invasion of host cells has been generated. These parasites, Mic1-3-KO, have been shown to reduce the incidence and severity of congenital transmission of OF1 mice challenged with the 76K strain of T. gondii [75]. Immunity was associated with enhanced maternal Th1 responses, specifically increased
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IFN␥ production by tachyzoite antigen stimulated splenocytes from those mice which had been previously vaccinated with Mic1-3-KO compared with non-vaccinated control mice. However, IL-10 levels were also increased in the vaccinated mice, which might be significant in preventing abortion or foetal death. 8. Conclusions The murine model has proven useful for understanding congenital toxoplasmosis, but also some limitations for studying congenital toxoplasmosis in humans. In particular the BALB/c mouse has proven particularly useful as this strain of mouse can develop immunity capable of preventing congenital disease transmission as observed in humans. The murine model has been employed to dissect the role of parasite multiplication and the immune response on foetal death and disease transmission for vaccination. It has also been effective to test the ability of chemotherapeutics to limit vertical disease transmission [80]. The relatively short gestation time facilitates reasonably efficient experimentation, but is also a limitation. Specifically, it is challenging to study the effect of immune modulation in the third trimester of pregnancy on parasite multiplication or vice versa. However, the availability of numerous immunological reagents and the continued expansion of the catalogue of gene deficient mice will ensure its continued utility and should provide increased understanding of the pathogenesis of congenital T. gondii infection. References [1] Dubey JP. Sources of Toxoplasma gondii infection in pregnancy. BMJ 2000;321:127–212. [2] Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004;363:1965–76. [3] Sensini A. Toxoplasma gondii infection in pregnancy: opportunities and pitfalls of serological diagnosis. Clin Microbiol Infect 2006;12:504–12. [4] Dunn D, Wallon M, Peyron F, Peterson E, Peckham C, Gilbert R. Mother-tochild transmission of toxoplasmosis: risk estimates for clinical counselling. Lancet 1999;353:1829–33. [5] Wallon M, Liou C, Garner P, Peyron F. Congenital toxoplasmosis: systematic review of efficacy of treatment in pregnancy. BMJ 1999;318:1511–4. [6] Tenter AM, Heckeroth AJ, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2000;30:1217–58. [7] Pelloux H, Brenier-Pinchart MP, Fricker-Hidalgo H. Protozoan infections in humans: congenital toxoplasmosis. Eur J Protistol 2003;39:444–8. [8] Bennett R, Christiansen K, Clifton-Hadley R. Preliminary estimates of the direct costs associated with endemic diseases of livestock in Great Britain. Prev Vet Med 1999;39:155–71. [9] Guerina NG, Hsu HW, Meissner C, Maguire JH, Lynfield R, Stechenberg B, et al. Neonatal serologic screening and early treatment for congenital Toxoplasma gondii infection. N Engl J Med 1994;330:1858–63. [10] Roberts CW, Alexander J. Studies on a murine model of congenital toxoplasmosis: vertical disease transmission only occurs in BALB/c mice infected for the first time during pregnancy. Parasitology 1992;104:19–23. [11] Beverley JK. Congenital transmission of toxoplasmosis through successive generations of mice. Nature 1959;183:1348–9. [12] Remington JS. Experiments on the transmission of toxoplasmosis. Surv Ophthalmol 1961;6:856–76. [13] De Roever-Bonnet H. Congenital toxoplasma infections in mice and hamsters infected with avirulent and virulent strains. Trop Geogr Med 1969;21:443–50.
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