The gonads and the thymus

Simple experimental approaches, like neonatal gonadectomy and thymectomy, and/or whole body irradiation of mice previous to cysticercosis infection, suggest that both the endocrine and immune systems are involved in the hosts' sexual differences of parasite loads, when measured 30 days after infection. Orchidectomy greatly increases parasite loads while ovariectomy reduces them (Huerta et al. 1992). Oestradiol and testosterone supplementation of gonadectomized mice restores their parasite loads to normal levels (Huerta et al. 1992; Terrazas et al. 1994). Thymus function seems to hinder parasite numbers in both sexes but more so in males than in females and, thus, sexual differences tend to disappear in thymectomized mice (Bojalil et al. 1993). Likewise, irradiation favours parasite growth in both male and female mice but more so in the male hosts (Huerta et al. 1992). T cell replenishment in thymectomized male and female mice, but not passively administred mouse whole immune sera, restores parasite loads and sexual differences in favour of females in association with restoration of cutaneous delayed hypersensitivity reactions (Bojalil et al. 1993). Vaccination is more effective in male mice than in female mice, both in reducing parasite loads and in increasing the number of mice totally free of parasites (Sciutto et al. 1990). Furthermore, a Th1 response is involved in resistance to infection, whereas Th2 activity associates with heavy parasite intensities, since mice that received anti-interleukin 10 (IL-10) Ab or recombinant IL-10 carried lower parasite intensities and developed a strong Th1-type response, while mice receiving anti-interferon gamma (IFN-γ) and anti-interleukin-2 (IL-2) Ab's, or recombinant IFN-γ or recombinant IL-2 showed a dramatic increase in susceptibility. Thus, the Th1-type immune response plays a fundamental role in protection against Taenia crassiceps, whereas Th2 does nothing, or may favour parasite establishment (Terrazas et al. 1999).

Together, the above findings prompt the hypothesis that sex steroids act upon parasite loads: oestradiol increases and testosterone limits parasite loads by mechanisms that possibly involve both the immune system of the host and the parasites reproductive system.

The endocrine-immune connection: do sex steroids control the host's immune response in cysticercosis?

The simplest hypothesis to connect the immune response with the females' greater parasite load is to propose that oestradiol directly favours a parasite-permissive Th2 immune response, which in turn down-regulates a parasite-hindering Th1 response. Thus, parasites grow immunologically undisturbed in a female endocrinological environment. The first premise is in keeping with the general notion that females produce more antibodies than males (Ahmed, Talal and Christadoss, 1987; Roberts et al. 2001) and the second refers to Th1/Th2 mutual downregulation (Mosmann and Coffman, 1989).

However, the numbers and types of lymphoid cells trafficking inside the infected peritoneal cavity do not show sexual differences in infected mice (Padilla et al. 2001), nor do the levels of circulating specific antibodies (Sciutto et al. 1990) or splenocyte proliferation and cytokine synthesis in response to in vitro antigen stimulation (Terrazas et al. 1998). The sera of infected mice of both sexes reflect a significant shift from Th1 to Th2 immune response during the course of infection. This is characterized by a marked decrease of IL-2 and IFN γ, enhancement of IL-6, IL-10 and IL-4 secretion (Terrazas et al. 1998; Toenjes et al. 1999; Rodríguez-Sosa et al. 2002). The shift starts in the first week of infection, when parasite reproduction rates are low, reaching an equilibrium in favour of Th2 at 4–6 weeks of infection, when parasites reproduce vigorously. However, the Th1/Th2 shift occurrs similarly in both male and female mice.

Thus, a major sex-associated immunological difference in antibodies or cytokines during infection that would account for the greater parasite loads in female mice could not be established.

The immuno-endocrine connection: the role of the IL6 and P-450 aromatase

(3) The molecular mechanisms of feminization

Feminization of chronically infected male mice logically follows from the IL-6's known capacity to induce the expression of P-450 aromatase (Reed et al. 1993). This enzyme's overexpression in the male host can shunt the metabolic pathway of testosterone towards dihydrotestosterone and switch it to oestradiol and, hence, feminization, TH1 inhibition and parasite replication. Fig. 1 depicts this hypothetical regulation network. Studies of changes in the infected mice mRNA levels for the enzymes involved in normal male steroid metabolism during infection reveals a decrease in the expression of 5α-reductase type II (the enzyme in charge of the conversion from testosterone to DHT) and an increase in the expression of P450-aromatase (responsible for the conversion of testosterone to oestradiol) (Morales-Montor et al. 1999). So, the shunt of testosterone to oestradiol is indeed turned-on in late infections and the metabolic pathway towards DHT is turned-off in chronically infected male mice. Feminization of the infected male has at least one plausible mechanism mediated by the immune system via Th2-dependent IL-6 (Fig. 1).

Fig. 1. Chart of the immunoendocrine mechanism of feminization in chronically infected male mice. The cysticercus antigenically stimulates the immune system: TH2 cells produce IL-6 in addition to other molecules. IL-6 in turn, stimulates the expression of the enzyme P-450 aromatase, which aromatizes testosterone (Te) to oestradiol (E2), instead of its conversion to dihydrotestosterone (DHT) by 5α-reductase. Thus, with time of infection, Th2 is progressively reinforced and Th1 is inhibited. In consequence, the infected male becomes feminized (oestrogenized and deandrogenized), favouring cysticercus reproduction because the high E2 endocrine environment preferentially stimulates Th2 over Th1.

The suggestion that the immune system is indeed involved in feminization is supported by the finding that newly-irradiated or thymectomized male strain C57/Bl6 mice fail to feminize when chronically infected (Morales-Montor et al. 2001). The role the of IL-6 is further strengthened by the fact that IL-6^-/- (KO) infected mice do not develop the feminization process, while the restitution with IL-6 restores feminization. Further involvement of IL-6 is documented by the testes-enhanced expression of the IL-6 gene. IL-6 has been shown to activate aromatase expression in the testes of cysticercotic mice and to induce active aromatization from androgens to oestrogens (Morales-Montor et al. 2001; Morales-Montor et al. 2002a,b,c). The increased serum levels of follicle-stimulating hormone (FSH) (the natural activator of aromatase expression, the secretion of which is also influenced by IL-6) detected in chronically infected mice, supports the notion that FSH interaction with IL-6 in the testes may cooperate in the induction of the feminization process (Morales-Montor et al. 2001).

(6) The involvement of thymus and spleen in feminization

The question as to whether oestrogenization and deandrogenization affect the immunologically prominent thymus and spleen has been studied by mRNA expression patterns of c-fos and c-jun, two estradiol-regulated genes, as well as those of the p53 and bcl2 genes in the testes, spleen, and thymus of infected males. It was found that c-fos was significantly increased in all organs, c-jun was increased only in the thymus, p53 mRNA was markedly reduced in all tissues, and bcl2 was abolished only in the thymus (Morales-Montor et al. 1998). Thymic cell analysis performed by flow cytometry showed a reduction in the content of the CD3+, CD4+, and CD8+ subpopulations in the infected male mice. This finding suggests that the increase in oestradiol levels of the host could change the expression pattern of several genes that participate in the regulation of apoptosis in the thymus of male mice during chronic infection with T. crassiceps cysticerci, and that oestrogens may inhibit the cellular immune response to the parasite (Morales-Montor et al. 1998).

In an effort to identify the sex steroids involved in this gene scenario, the effects of testosterone, dihydrotestosterone, and 17β-estradiol in castrated mice of both sexes infected with Taenia crassiceps cysticerci were studied. Castration and treatment with either testosterone or dihydrotestosterone before infection markedly decreases parasite loads in both genders, while the treatment with 17β-oestradiol increases it in both genders. The specific splenocyte cell proliferation and IL-2 and IFN-γ production are depressed in infected-castrated mice of both genders, while treatment with testosterone or dihydrotestosterone produces significant cell proliferation recovery and enhanced production of IL-2 and IFN-γ. The opposite effect of the same sex steroids is found in the humoral response machinery: it is unaffected by testosterone or dihydrotestosterone restitution, while the treatment with oestradiol of both genders augmented the levels of anti-cysticercus IgG, and of IL-6 and IL-10 production. These results are congruent with the idea that androgens mediate immune functions which limit the cysticercus load, possibly through the stimulation of specific cellular immunity of the host (Morales-Montor et al. 2002a,b) (Fig. 2).

Fig. 2. Chart of the host-parasite neuroimmunoendocrine network in the control of cysticercus reproduction. Sex steroids may act directly upon the parasite whereby progesterone (P4) and oestradiol (E2) favour its reproduction, and dihydrotestosterone (DHT) inhibits it. Also, sex steroids act upon parasite loads via the immune system, E2 favouring a permissive Th2 response that inhibits the restrictive Th1. The central nervous system (CNS) of the host shows changes that reveal that it senses and may react to the infection. The arrows show the interconnection among all system components. (+) Positive stimulation; (−) negative stimulation.

Direct action of sex steroids upon the cysticercus

Sex steroids can regulate parasite loads through their reciprocal interaction with immune mechanisms but, in addition, they can act directly upon the cysticercus reproductive system (Fig. 2). Oestradiol, and progesterone to a lesser extent, when added to in vitro cultures of T. crassiceps ORF strain, stimulate the parasite's production of buds, their DNA synthesis and [³ H]thymidine uptake, while testosterone and DHT are slightly inhibitory and even exert a pathogenic effect on the parasites (Escobedo et al. 2005). The possible molecular mechanisms by which sex-steroids affect T. crassiceps reproduction imply the presence in the parasite of hormone receptors, a totally unknown issue in cestodes. RT-PCR, and sequencing the specific amplified fragments, have shown the presence of oestradiol receptors (both α and β isoforms) and an androgen receptor, but not of progesterone receptors in the cysticerci of T crassiceps. Moreover, morphological studies of the parasite after sex-steroid treatment demonstrated that the E₂ and P₄-treated cysticerci are quite mobile, transparent, and surrounded by abundant buds. In contrast, T₄ and DHT-treated cysticerci are smaller than the E₂ or P₄-treated or than untreated cysticerci, and undergo progressive internal disorganization and develop opaque, whitish masses in their teguments, as well as showing progressive loss of motility. Furthermore, not only in vitro treatment of cysticerci affected their survival, but also pre-treatment of T. crassiceps with sex-steroids prior to their inoculation in male or female recipient mice had an effect on their infectivity: oestrogens promoted and androgens inhibited their infectivity. In contrast, prior exposure of parasites to T₄ and DHT significantly decreased the expected parasite load in male and female hosts (Escobedo et al. 2005). These last results are the first demonstration that sex steroids of the host may act directly upon parasite reproduction by binding to classic and specific sex-steroid receptors on the parasite and stimulating their natural functions of controlling infectivity, differentiation, reproduction and apoptosis (Escobedo et al. 2005).

Furthermore, it has been shown that T. crassiceps cysticerci are able to metabolize sex-steroid precursors, and thus the parasites can make their own sex-steroids and are possibly sensitive to the host's regulators of hormonal expression (Gómez et al. 2000).

Hosts' behavioural changes in the infected host

The changes in hormonal host environment in male mice induced by T. crassiceps infection also affect other physiological aspects of the host, such as sexual (Morales et al. 1996), aggresive (Gourbal, Lacroix and Gabrion, 2002) and territorial behaviours (Gourbal et al. 2001). Male mice infected with T. crassiceps show remarkable changes in sexual behaviour: mounting, intromission, and ejaculation responses markedly decline as infection progresses. These sexual changes are characterized by a complete loss of the ejaculation response early at infection (6 weeks), followed by a gradual decrease in the number of mounts and intromissions, and their latencies were increased, until none of the parasitized mice showed any sexual response toward female mice (Morales et al. 1996). Moreover, changes in the sexual behaviour of infected male mice were associated with the serum levels of their sex-steroids, and testosterone or dihydrotestosterone restitution completely restored their normal sexual behaviour (Morales et al. 1996). Furthermore, experimental infection with T. crassiceps cysticerci disrupted the dominant-subordinate status (Gourbal et al. 2002). In infected male mice strong perturbations in territorial behaviour and aggressiveness are found. Infected dominant male mice do not show a significant reversal of dominance order compared to uninfected mice, and during the confrontation between naive infected and healthy mice, infected animals more often assume a subordinate status than healthy mice. The effects of infection by T. crassiceps were more likely to prevent adult male mice from becoming behaviourally dominant than to reverse existing dominance relationships (Gourbal et al. 2001, 2002).