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Introduction
Chagas disease (CD) is a parasitic zoonosis caused by the protozoan Trypanosoma cruzi. Even this disease is endemic of America, nowadays, is considered an emerging worldwide disease affecting nearly 8 million people. CD incidence is 30,000 cases yearly, and currently, is the most lethal parasitosis with 14,000 deaths annually1.
As Chagas himself said "the virulence of Schizotrypanum cruzi (now Trypanosoma cruzi) is influenced by several factors, whose nature and effects we do not know yet"2. Currently, the factors that determine host susceptibility to T. cruzi infection remained unknown.
The host endocrine system plays an important role in the establishment and development of the parasitic infections, importantly the sexual dimorphism (different host susceptibility between males and females)3. It has been described that the testosterone (T) increases host susceptibility and decreases the immune system efficiency4 and, the estradiol (E2) has the opposite effect5.
In another parasitosis, sexual dimorphism was reported. In leishmaniasis, male-infected hamsters had bigger lesions than females6, and men are infected more often than women7. Hence, better understanding of sexual dimorphism in parasitic diseases will allow the development of more efficacious treatments7.
Regarding to CD, it has been proposed that the microenvironment of each sex modulates the host response. However, very little is known about it.
Materials and methods
Parasites and animals
T. cruzi NINOA strain, member of the predominant lineage in Mexico, was used in these experiments8.
Adult female and male BALB/c mice (6 weeks old) were maintained and handled according to the Animal Care and Use Committee Guide9. The protocol for this study was evaluated and approved by the Bioethics Committee.
Mice were divided into six animal groups as follows: uninfected or infected intraperitoneally with 1 × 103 trypomastigotes10 (infected, sham infected, orchiectomized, orchiectomized and supplemented with estradiol, orchiectomized supplemented with testosterone, oophorectomized, oophorectomized and supplemented with estradiol, and oophorectomized and supplemented with testosterone). The sham group were mice that had a simulated operation, they were opened and closed without any organ removal, just to verify if the operation istelf causes any alteration.
Gonadectomy
Oophorectomies and orchiectomies were performed on mice. In addition, fake operations (sham) were performed to account any change that the operation itself could had in T. cruzi infection behavior.
Hormones administration
Estradiol (5 mg/kg) (Whitney, Mexico City, Mexico) and testosterone (20 mg/kg) Ara-Test 2500 (Laboratorios Aranda, Mexico City, Mexico) were administrated subcutaneously weekly throughout the experiment (3 months).
Clinical features, parasitemia, and mortality
Clinical features presented during the parasitemia curves were recorded. Parasitemia was quantified according to the modified Brennerʼ technique employing a Neubauer chamber Blaubrand (Merck, Darmstadt, Germany). Then, parasitemia curves were constructed11. Mice death was recorded daily. All experiments were performed in triplicate.
Histopathological analysis
In the parasitemia peak (the day with the most parasites), mice were sacrificed12. Then, hearts were obtained, paraffin embedded, and blocks were sliced on a microtome Thermo Scientific HM325 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) to obtain 30 sections of 5μm (6 slides per heart), slides were stained with the hematoxylin/eosin (HE) technique. For each section, six microscopic fields were counted through a microscope Nikon-Eclipse 50i (Nikon, Chiyoda, Japan) at a magnification of ×40. Amastigote nest numbers were recorded. Inflammatory infiltrate was ranked as follows: absent (−), mild (< 25% of the field), moderate (25-50% of the field), and severe (> 50% of the field).
Results
Clinical features, parasitemia, and mortality
Since the second week post-inoculation (p.i.), parasitemia was positive. On the third week p.i., infected mice presented: piloerection and postration; paresia was presented only in higher estradiol groups. Statiscally, T. cruzi-infected and sham T. cruzi-infected groups behaved similarly (both sex groups) (p < 0.05). Regarding to the females, the oophorectomized T. cruzi-infected group had the highest parasitemia peak (1.75 x 105 parasites/mL) at day 17 p.i. contrasting ooporectomized and estradiol supplemented T. cruzi-infected group has the lowest (5 x 104 parasites/mL) at day 24 p.i. on the other hand, concerning to malesthe highest parasitemia peak was presented by T. cruzi-infected and T-cruzi sham infected groups (4.8 x 105 parasites/mL) at day 33 p.i. whereas the lowest peak was presented by orchiectomized (Fig. 1).
Figure 1 Parasitemia curves of the behavior of Trypanosoma cruzi in mice of each mice group among all the experiment; the lines are discontinued if the mice group died. A: females. 1. Uninfected (black). 2. T. cruzi-infected (green). 3. Sham T. cruzi-infected (purple). 4. Oophorectomized T. cruzi-infected (red). 5. Oophorectomized and supplemented with estradiol T. cruzi-infected (pink). 6. Oophorectomized and supplemented with testosterone T. cruzi-infected (blue). B: males. 1. Uninfected (black). 2. T. cruzi-infected (green). 3. Sham T. cruzi-infected (purple). 4. Orchiectomized T. cruzi-infected (red). 5. Orchiectomized and supplemented with estradiol T. cruzi-infected (pink). 6. Orchiectomized and supplemented with testosterone T. cruzi-infected (blue).
To compare the parasitemia, the area under the curve was calculated. Regarding females, oophorectomized T. cruzi-infected group presented the highest parasitemia whereas oophorectomized supplemented with estradiol T. cruzi-infected group, the lowest. In males, the highest parasitemia was displayed by the T. cruzi-infected, while orchiectomized supplemented with estradiol T. cruzi-infected group, the lowest. Comparing both sexes, T. cruzi-infected and sham T. cruzi-infected groups had a similar parasitemia, showing that the fake operation does not change the infection course by itself. The highest parasitemia corresponded to T. cruzi-infected and sham T. cruzi-infected males; oophorectomized supplemented with estradiol females had the lowest (Fig. 2).
Figure 2 Parasitemia presented in Trypanosoma cruzi-infected mice. Males (black) and females (gray). 1. Uninfected, 2. T. cruzi-infected. 3. Sham T. cruzi-infected. 4. Gonadectomized T. cruzi-infected. 5. Gonadectomized and supplemented with estradiol T. cruzi-infected. 6. Gonadectomized and supplemented with testosterone T. cruzi-infected.
Mortality percentage was higher in males than in females. Regarding to females, deceases were present mainly in the oophorectomized, stradiol supplemented, T. cruzi-infected group; whilst in minor amount in the oophorectomized T. cruzi-infected group. In contrast, males died on almost all groups (except on the orchiectomized supplemented with estradiol T. cruzi-infected group). The highest mortality was presented by orchiectomized supplemented with testosterone T. cruzi-infected group (Fig. 3).
Figure 3 Mortality of the Trypanosoma cruzi-infected mice. Males (black) and females (gray). 1. Uninfected. 2. T. cruzi-infected. 3. Sham T. cruzi-infected. 4. Gonadectomized T. cruzi-infected. 5. Gonadectomized and supplemented with estradiol T. cruzi-infected. 6. Gonadectomized and supplemented with testosterone T. cruzi-infected.
Histopathological analysis
Hearts of T. cruzi-infected mice showed cardiomegaly. Males showed higher cardiomegaly rate, ascites, and hepatomegaly; females showed more edema and necrosis.
Oophorectomized T. cruzi-infected group had more amastigote nests, whilst, orchiectomized supplemented with testosterone T. cruzi-infected group had severest inflammatory infiltrate. Orchiectomized T. cruzi-infected group had less amastigote nests, and orchiectomized supplemented with estradiol T. cruzi-infected group had less inflammatory infiltrate (Fig. 4).
Figure 4 Microscopic analysis of the heart tissues of Trypanosoma cruzi-infected mice. Heart tissues were stained according to the HE technique to show the T. cruzi amastigote nests NINOA strain, edema, and the inflammatory infiltrate. The photograph was taken at ×40 magnification. A: healthy mice tissue. B: heart tissue T. cruzi-infected female mouse, amastigote nest, and mild acute inflammatory infiltrate. C: heart tissue T. cruzi-infected male mouse, amastigote nests, edema, and severe acute inflammatory infiltrate. Amastigote nest (white arrow); inflammatory infiltrate (black arrow).
Discussion
The host-parasite relationship is multifactorial; sex hormones could affect infection intensity13. Sexual dimorphism has been scarcely reported for CD14.
Clinically, infected mice from both sex groups presented piloerection caused by adrenergic receptor activation in stressed individuals and prostration that has been described as acute phase CD sign on humans with T. cruzi-infection14. Both features have been reported with T. cruzi Y strain5, as well as the paresia that leads them to take a hunch posture15, presented in the mice groups with higher E2 concentration. This could be explained by host autoantibodies denervation, as occurred in the Chagasic hearts16.
In males, orchiectomized group presented fewer parasites than non-orchiectomized ones as was reported in T. cruzi Y strain5. In females, higher E2 groups had less parasitemia as has been reported17.
Heart failure implies cardiomegaly, accompanied by hepatomegaly, ascites, and edema18, signs presented in the T. cruzi-infected mice. Hepatomegaly is a consequence of this organ´s hypoperfusion, in fact, ascites accompanies this sign on 25% of the cases19. Inflammation causes edema20 and necrosis, resulted from ischemia produced by the reactive oxygen species (ROS) released in the inflammatory process.
In terms of mortality, higher T concentration correlated with a higher mortality rate accordingly with Tulahuén strain T. cruzi-infected reports21. In CD Mexican reports, most of the patients are women13. It has been proposed that the parasites have a preference to infect the sex that lives longer22; further, we propose that men, due to the higher death rates (curiously related to cardiovascular diseases), die before a proper CD diagnosis could be done.
Higher T concentration groups presented more severe inflammatory infiltrate; accordingly, cardiac damage may be a consequence more than the presence of the parasite itself, of the inflammatory oxidative damage21. Besides, T. cruzi infection disturbed mitochondrial membrane, leading to ROS production, that oxidize lipids, proteins, and deoxyribonucleic acid (DNA)23.
In this regard, estrogens are a cardiovascular protector factor because they can decrease the NO (potent vasodilator) concentration21, helping the host. In contrast, the male immune system could be weaker due to the oxidation handicap hypothesis (OHH), which postulates that males have a higher T concentration, implying an overproduction of ROS, making them more susceptible to tissue damage24.
Moreover, host´s cells mitochondrial antioxidant function is lower in males23, and antioxidant enzymes, superoxide dismutase and catalase, concentrations are host sex-related (83% males versus 180% females)25.
It has been reported that T concentration does not influence the severity of the cardiac damage26 contrasting with the present study where T does influence it.
Thus, in CD, inflammation plays a main role in tissue damage, and in patients´ death, because this protozoan avoids the oxidant action, but the host cell does not27. Sex hormones have been related to this because can control the macrophages21; the feminine supremacy paradigm stablishes that the female hosts are more resistant to parasitosis because they have a different immune response17; for example, macrophages have estrogen receptors (ERα) that modulate the oxidative microenvironment28. Moreover, E2 inhibits M1 macrophage activation (by inhibiting the metalloprotease 9)29 having an anti-inflammatory activity, but leads to the interleukin 10 production and to the chronicity of the infection30.
Notwithstanding, as T. cruzi affects cytokine release in the host, this parasite could modulate macrophage´s polarization in CD and this process could be sex hormone mediated. Therefore, experiments are being carried out in this regard.
Conclusions
The data presented showed that testosterone increased infection severity, whereas estradiol had the opposite effect, not only confirm previous data but also expanding and detailing these phenomena, highlighting the importance of T. cruzi host sex bias. This research improves the understanding of sex hormones´ infuence upon this infection to contribute with the handling of Chagas disease.
