Keywords:
neurocysticercosis - neglected diseases - Taenia
Palavras-chave:
Neurocisticercose - doenças negligenciadas - Taenia
Taeniasis is the infection by the adult worm of the Taenia spp., while cysticercosis
is the infection with the larval form of the parasite. Human cysticercosis is caused
by the presence of T. solium cysticerci[1]. The World Health Organization considers both diseases to have been neglected[1],[2]. The taeniasis/cysticercosis complex is a prominent cause of chronic debilitating
illness, morbidity and mortality[3], with worldwide prevalence, especially in rural communities of developing countries[2].
Neurocysticercosis (NCC) is the most frequent parasitic infection of the central nervous
system (CNS) and the main preventable cause of acquired epilepsy[4]. Up to 50% of the NCC cases are asymptomatic. The symptoms vary from seizures, headaches,
focal neurologic deficits, cognitive decline and intracranial hypertension. The clinical
manifestations depend on factors related to the parasite, such as size, location,
and development stage, or factors related to the host, such as the immune response[4],[5],[6]. The great variety of clinical forms are related to the modulation of the immune
profile due to the presence of viable cysticerci in the brain (asymptomatic NCC),
or to an intense inflammatory response (symptomatic NCC)[7],[8],[9].
Usually, when an organism is exposed to a pathogen it generates an immunologic response
aimed at the pathogen's destruction. The protective or pathogenic efficacy of this
innate immune response greatly depends on the generation of unspecific inflammatory
phenomena in the surroundings of the pathogen. The adaptive immune response relies
on the selective clonal proliferation of systemic lymphoid cells followed by a differentiation
of effective Th1, Th2, Treg and Th17 cells, among other profiles that induce cytokine
or antibody production[5],[6],[7],[8],[9].
The presence of the viable T. solium cysticercus in the brain tissue of humans triggers the increase of local inflammatory
cells through the main pathways of leukocyte infiltrations, which are the parenchymal
vessels, subarachnoid space and choroid space[10]. Within the CNS, the parasite may install itself in the brain parenchyma, subarachnoid
space, ventricular system[11] and, less frequently, it can be found in the brainstem[12] and in the spinal cord[13].
One of the experimental models used to study T. solium cysticercosis is T. crassiceps due to their epitope similarity[14]. The immune response in mice infected with T. crassiceps is not well described in NCC models. However, Moura et al.[15], studying an intraventricular experimental NCC model, reported that BALB/c mice
presented with a predominance of a systemic mixed Th1/Th17 immune profile in the initial
phases of the infection, while this gradually modified into a Th2 profile through
the course of the infection.
Currently, there are five different experimental models used for NCC studies. The
first one developed by Cardona et al.[7], used BALB/c female mice infected with Mesocestoides corti. Subsequently, our group found that the intracranial inoculation of T. crassiceps cysticerci in both BALB/c and C57BL/6 mice lineages showed the susceptibility and
resistance of different lineages[16]. Other examples are the inoculation of active T. solium oncospheres in rats[17] and the inoculation of active T. solium oncospheres in the subarachnoid space of young swine[18]. The most recent model described simulated the extraparenchymal racemose NCC in
rats through the inoculation of T. crassiceps cysticerci[19].
It is important to highlight that different mice lineages produce different inflammatory
responses with different cellular and cytokine profiles[20]. The subcutaneous inoculation of T. crassiceps cysticerci allowed analysis of the kinetics of the infection in susceptible (BALB/c)
and resistant (C57BL/6) mice lineages, showing a greater parasitic burden in the susceptible
mice[21].
Other studies of NCC models[16] and in a subcutaneous model[22] showed that BALB/c mice are less efficient in the precocious destruction of the
parasite, allowing its proliferation with greater inflammatory intensity in the acute
phase of the infection, with a predominance of polymorphonuclear cells and macrophage
infiltration. The C57BL/6 mice lineage is capable of inducing the parasite death with
greater inflammatory intensity in the late phase of the infection with less tissue
injury and a predominance of infiltration of mononuclear inflammatory cells. These
findings confirmed the resistance profile of the C57BL/6 mice lineage in both experimental
models.
To date, there is little description of the brain lesions found in the resistant mice
lineage (C57BL/6) in experimental NCC, and no reports on the local immune response
in this kind of infection. Therefore, the aim of this study was to describe the brain
injuries and the in situ immune response in C57BL/6 mice inoculated intracranially with T. crassiceps cysticerci.
METHODS
Parasite maintenance
This study was approved by the Ethics Committee in Animal Use of the Federal University
of Goiás, protocol number 065/2015. The maintenance of the T. crassiceps ORF strain was performed through successive intraperitoneal passages every 90 days
in BALB/c female mice aged 8–12 weeks old[14]. The parasite is maintained in the animal facilities of the Tropical Pathology and
Public Health Institute of the Federal University of Goiás.
Infection and euthanasia of the mice
Wild type C57BL/6 female mice aged 8–12 weeks old, weighing between 20–30 grams were
used. They were divided into a control (CT) group and an infected (INF) group. Mice
from the INF group were weighed, intraperitoneally anesthetized with ketamine (100
mg/mL) and xylazine (20 mg/mL) in a ratio of 0.1 mL/10g. Afterwards, the animals were
inoculated intracerebrally as described previously[16]. The CT group (n = 9) received an inoculation of physiologic solution (NaCl 0.9%)
in a similar volume to the INF group (n = 15).
Ninety days after the inoculation, all the mice were euthanized and had their brains
removed.
Histopathology analysis
The histopathology analysis was performed in fragments of the brain tissue using a
matrix for histological slices (Insight®), fixed in a solution of 4% paraformaldehyde and 70% alcohol, dehydrated with alcohol,
diaphonized in xylol, and embedded in paraffin. The blocks were cut into 4 μm width
slices. The fragments were captured with glass slides and stained with hematoxylin-eosin[15],[16].
The general pathologic processes were described and classified in a semi-quantitative
analysis as follows: absent when there was no compromise of the host tissue, score
= 0; discrete with up to 25% of area commitment, score = 1; moderate from 26% to 50%
of area commitment, score = 2; and accentuated with more than 50% of area commitment,
score = 3[15],[16].
Immunoenzymatic assay
After the euthanasia, a fragment of the brain tissue was frozen at -80ºC for the enzyme-linked
immunosorbent assay (ELISA) analysis. After this it was sonicated with a solution
of Tris HCl, NP40 1% (SIGMA) and protease inhibitor[23].
The interleukin (IL)-4, IFN-gamma and IL-10 cytokines were quantified in the brain
tissue homogenate solution through sandwich ELISA according to the manufacturer's
instructions (BD OptEIA™). The IL-4 and IL-10 dosages were performed with Mouse IL-4 ELISA (Lot: 26389) and Mouse IL-10 ELISA (Lot: 20169) BD OptEIA™ Sets (BD Biosciences, San Diego, CA). The IFN-gamma
dosage was performed with ELISA high binding microplates sensitized with 80 μL of
IFN-gamma monoclonal antibody (5μg/mL of clone XMG 1.2 in PBS). The optic density
was detected through the Thermo/Labsystems microplate reader, using specific filters
for each cytokine[23].
Statistical analysis
The statistical analysis of the histopathology was performed using the Sigma Stat
2.3 software. The Mann-Whitney test was used, followed by the Bonferroni post-test.
Differences were considered significant when p < 0.05.
The statistical analysis of the in situ cytokine dosage was performed with the GraphPadPrism 5.0 software, and after the
establishment of distribution and variance of the samples, the T-test was used. Differences
were considered significant when p < 0.05.
RESULTS
The histopathology analysis allowed the evaluation of the CNS infection of C57BL/6
mice with viable T. crassiceps cysticerci. The general pathologic processes found are described in the [Table]. The INF group had a greater degree of meningitis, parenchymal edema, mononuclear
inflammatory cell infiltration and hemisphere compression (p < 0.05) than the CT group
([Figures 1] and [2]).
Table
General pathologic processes found in C57BL/6 mice inoculated intracranially with
viable Taenia crassiceps cysticerci.
|
Pathologic processes
|
CT group Median (min.-max.)
|
INF group Median (min.-max.)
|
p
|
|
Meningitis
|
0 (0–0)
|
0 (0–1)
|
0.012*
|
|
Parenchymal edema
|
0 (0–0)
|
1 (0–3)
|
0.011*
|
|
Hyperemia
|
0 (0–0)
|
0 (0–1)
|
0.176
|
|
Perivasculitis
|
0 (0–0)
|
0 (0–2)
|
0.177
|
|
Inflammatory infiltration (MN)
|
0 (0–0)
|
0 (0–2)
|
0.008*
|
|
Microgliosis
|
0 (0–0)
|
0 (0–2)
|
0.106
|
|
Perivascular edema
|
0 (0–0)
|
0 (0–1)
|
0.491
|
|
Ventriculomegaly
|
0 (0–0)
|
0 (0–3)
|
0.177
|
|
Hippocampus alterations
|
0 (0–0)
|
0 (0–3)
|
0.291
|
|
Choroiditis
|
0 (0–0)
|
0 (0–0)
|
1
|
|
Ependymitis
|
0 (0–0)
|
0 (0–0)
|
1
|
|
Tissue loss
|
0 (0–0)
|
0 (0–3)
|
0.108
|
|
Hemorrhage
|
0 (0–0)
|
0 (0-0)
|
1
|
|
Hemisphere compression
|
0 (0–0)
|
2 (0–3)
|
0.006*
|
CT: control group; INF: infected group; MN: mononuclear cells; *statistically significant
differences (p < 0.05).
Figure 1 Photomicrograph of the general pathologic processes found in C57BL/6 mice inoculated
intracranially with viable Taenia crassiceps cysticerci after 90 days. A. Discrete meningitis in the infected (INF) group (arrow)
(scale = 100 μm); B. Accentuated parenchymal edema in the INF group (arrow) (scale
= 100 μm); C. Moderate perivasculitis in the INF group (arrow) (scale = 50 μm); D.
Moderate microgliosis in the INF group (arrows) (scale = 100 μm). H&E.
Figure 2 Mesoscopic image of the brains from C57BL/6 mice. A. Absence of hemisphere compression
in a mouse from the control (CT) group. B, C and D. Hemisphere compression, dislodgement
of the medial line, hippocampus and lateral ventricle in mice inoculated intracranially
with Taenia crassiceps cysticerci (INF group). In D, the arrow shows the presence of a viable cysticercus
in the lateral ventricle. Scale bar = 1 mm. H&E.
The compression of the brain hemispheres with consequent dislodgement of the medial
line due to the installation and growth of the cysticerci was observed in the INF
group, both in the corticomeningeal region as well as in the lateral ventricle regions
([Figure 2]). These injuries were not observed in the control group.
The results of the in situ cytokine analyses are shown in [Figure 3]. It is possible to observe a significant increase in the IL-4 and IL-10 dosages
in the INF group when compared with the CT group (p < 0.05). There were no differences
in the IFN-gamma dosages between the two groups.
Figure 3
In situ cytokine dosage from C57BL/6 mice intracranially inoculated with Taenia crassiceps cysticerci, 90 days after the inoculation.CT: control group; INF: infected group.
*statistical difference between groups (p < 0.05), T Test.
DISCUSSION
This study showed the in situ histopathology and immune response of a resistant mice lineage (C57BL/6) given an
intracerebral inoculation of T. crassiceps cysticerci. The mechanisms in which the brain injuries are induced by the presence
of cysticerci depend on a combination of several factors, such as the site of implantation
of the parasite and the host immune-inflammatory response[6],[11], which is mediated by cytokines produced by resident and infiltrated cells, which
in turn are activated by the cysticerci antigens[4],[24].
The inoculated cysticerci were located in the ventricles or in the extraparenchymal/meningocortical
region and triggered a discrete inflammatory response with little mononuclear inflammatory
cell infiltration. As well, when the parasite installed in the surroundings of the
meninx, it was capable of inducing discrete meningitis. These findings are in accordance
with other reports on the location and inflammatory response of the T. crassiceps experimental model[7],[9],[16] as well as the human NCC inflammatory response[5],[11].
The histopathology findings described in this study are in accordance with Matos-Silva
et al.[16], who induced an experimental intraventricular NCC with T. crassiceps cysticerci in C57Bl/6 mice and described discrete mononuclear inflammatory cell infiltration,
parenchymal edema and meningitis. The findings described by Matos-Silva et al.[16] are similar to the those described in this study regarding the lineage, age, weight
and sex of the animals and the parasite used. However, in our study, the localization
of the parasite within the brain was mainly in the meningocortical region, differing
from the previously-cited study. This difference is important in the description of
the host response to the parasite, due to the fact that parenchymal NCC is associated
with seizures and focal neurological signs and is the form that presents with greater
inflammation when treated[26]. The human extraparenchymal NCC is associated with high mortality rates due to the
increase in intracranial pressure[6].
The hemispheric compression caused by the presence and growth of the cysticerci was
accentuated in the mice used in this study. The main consequence of this process is
the increase in the intracranial pressure which may lead to obstruction of CSF and
edema in different degrees of severity[11],[24]. The presence of parenchymal edema in the INF group of this study, even at 90 days
after the inoculation, may have been due to this compression process.
The in situ cytokine dosage results found in this study suggest the predominance of a Th2 immune
profile at 90 days after the inoculation of T. crassiceps cysticerci. This may be inferred by the significantly higher concentrations of IL-4
in the INF group when compared with the CT group and also because of the lack of difference
in the IFN-gamma concentrations. It is important to highlight that, within the brain,
the inflammatory and immune processes occur in a unique form, which may alter the
expected order of inflammation, tissue injury, damage to the host, death of the parasite
and resolution of the infection[24],[25].
The brain, as an immunoprivileged organ, attenuates the inflammation in its tissue
because it can threaten the brain's integrity and function[11],[24]. This protective mechanism is due to passive factors, such as the presence of the
blood brain barrier and the intracerebral circulation, as well as active factors such
as immunosuppressive cytokine production, neuropeptides, limitation in the MHC class
I and II expression, complement regulatory proteins and NK cell inhibitors, which
also contribute to the immune privilege[27],[28]. These factors act as a protective factor regarding NCC, preventing damage to the
brain tissue and favoring the extension of the parasite's life within the brain[29].
Moura et al.[15] reported that BALB/c mice, experimentally inoculated intracranially with T. crassiceps cysticerci, did not show significant differences in the in situ dosage of cytokines in spite of higher concentrations of IFN-gamma than IL-4, confirming
a Th1 immune profile, allowing the parasite survival. In our study, the C57BL/6 mice
inoculated with the same parasite showed a predominance of the Th2 immune profile
as an attempt to contain the parasite growth.
It is important to highlight the immunomodulation capacity of the cysticerci, which
is also responsible for the immune profile observed in C57BL/6 mice. The parasite's
antigens stimulate the production of antibodies and the release of chemical mediators
that are unable to destroy the cysticercus. This happens because the parasite simultaneously
modulates the action of these immune components and becomes viable for a longer period
of time[30]. Thus, it sways the immune profile from a Th1 response to a Th2 response, which
protects the tissue from damage, activates macrophages with lower microbicide potential,
and generates the activation and expansion of plasmocytes. As well, the IgE secretion,
presence of eosinophils, mastocytes and basophils are unable to destroy the parasite[6].
The IL-10 dosage detected in this study shows that the presence of this cytokine within
the brain tissue modulates the Th1 and Th2 immune responses. Interleukin-10 is a potent
inhibitor of the antigen presentation, and of the mediators and pro-inflammatory cytokine
production by macrophages, dendritic cells and TH1 cells[30]. It has an important regulatory role on the host inflammatory response during the
infection. The inflammation is essential at the beginning of the response against
the pathogen, but this must be controlled so it does not result in severe inflammatory
complications[31]. It is also capable of regulatory T cell lymphocyte activation, which decreases
the tissue damage[30]. Therefore, the statistically higher concentration of IL-10 in the infected group
in our study demonstrates that it is regulating the immune response in the mice, swaying
the immune response towards the Th2 immune profile, which decreases the parenchymal
damage and contributes to the parasite survival.
Therefore, when the immune aspects are correlated with the histopathology described
in this study, it is possible to conclude that the infection of T. crassiceps in C57BL/6 mice triggered an inflammatory response with a predominant Th2 and Treg
in situ response. These facts are reflected in the control of the intensity of the general
pathologic processes, such as meningitis, microgliosis, mononuclear inflammatory cell
infiltration and compression of the brain hemispheres.
