International Journal of
Molecular Sciences
Article
Role of the IL-33/ST2 Activation Pathway in the Development
of the Hepatic Fibrosis Induced by Schistosoma mansoni
Granulomas in Mice
Laura Maggi 1 , Genil Mororó Araújo Camelo 1 , Izabella Chrystina Rocha 1,2 , William Pereira Alves 3 ,
João Marcelo Peixoto Moreira 1 , Thiago Almeida Pereira 4 , Wagner Luiz Tafuri 5 , Élida Mara Leite Rabelo 3 ,
Ary Correa, Jr. 6 , Roselene Ecco 7 and Deborah Aparecida Negrão-Corrêa 1, *
1
2
3
4
5
6
7
*
Citation: Maggi, L.; Camelo, G.M.A.;
Rocha, I.C.; Pereira Alves, W.;
Moreira, J.M.P.; Almeida Pereira, T.;
Tafuri, W.L.; Rabelo, É.M.L.; Correa,
A., Jr.; Ecco, R.; et al. Role of the
IL-33/ST2 Activation Pathway in the
Development of the Hepatic Fibrosis
Induced by Schistosoma mansoni
Granulomas in Mice. Int. J. Mol. Sci.
2023, 24, 10237. https://doi.org/
10.3390/ijms241210237
Academic Editor: J. B. Helms
Received: 26 May 2023
Revised: 6 June 2023
Accepted: 13 June 2023
Published: 16 June 2023
Copyright: © 2023 by the authors.
Laboratório de Esquistossomose e Imunohelmintologia, Departamento de Parasitologia, Instituto de Ciências
Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil;
laauramaggi@gmail.com (L.M.); genilmororo@gmail.com (G.M.A.C.); izabella.bebel@hotmail.com (I.C.R.);
peixotomoreira@gmail.com (J.M.P.M.)
Curso de Enfermagem, Instituto de Ciências Biológicas e Saúde, Universidade Federal de Mato Grosso,
Barra do Garça 78698-000, MG, Brazil
Laboratório de Parasitologia Molecular, Departamento de Parasitologia, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil;
willip.alves@gmail.com (W.P.A.); rabelo@icb.ufmg.br (É.M.L.R.)
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine,
Stanford, CA 94305, USA; dealmeida.thiago@gmail.com
Laboratório de Patologia das Leishmanioses, Departamento de Patologia, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; wlrasotafuri@gmail.com
Laboratório de Micologia, Departamento de Microbiologia, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil; ary.correa.62@gmail.com
Setor de Patologia, Escola Veterinária, Universidade Federal de Minas Gerais,
Belo Horizonte 31270-901, MG, Brazil; eccoro.ufmg@gmail.com
Correspondence: denegrao@icb.ufmg.br; Tel.: +55-31-3409-2855
Abstract: Schistosoma mansoni eggs retained in host tissues induce innate cytokine release, contributing
to the induction of Type-2 immune responses and granuloma formation, important to restrain
cytotoxic antigens, but leading to fibrosis. Interleukin(IL)-33 participates in experimental models
of inflammation and chemically induced fibrosis, but its role in S. mansoni-induced fibrosis is still
unknown. To explore the role of the IL-33/suppressor of the tumorigenicity 2 (ST2) pathway, serum
and liver cytokine levels, liver histopathology, and collagen deposition were comparatively evaluated
in S. mansoni-infected wild-type (WT) and IL-33-receptor knockout (ST2−/− ) BALB/c mice. Our
data show similar egg counts and hydroxyproline in the livers of infected WT and ST2−/− mice;
however, the extracellular matrix in ST2−/− granulomas was loose and disorganised. Pro-fibrotic
cytokines, such as IL-13 and IL-17, and the tissue-repairing IL-22 were significantly lower in ST2−/−
mice, especially in chronic schistosomiasis. ST2−/− mice also showed decreased α-smooth muscle
actin (α-SMA) expression in granuloma cells, in addition to reduced Col III and Col VI mRNA levels
and reticular fibres. Therefore, IL-33/ST2 signalling is essential for tissue repairing and myofibroblast
activation during S. mansoni infection. Its disruption results in inappropriate granuloma organisation,
partly due to the reduced type III and VI collagen and reticular fibre formation.
Keywords: Schistosoma mansoni infection; liver fibrosis; granuloma; collagen deposition; reticular
fibres; IL-33/ST2 activation pathway
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Fibrotic diseases, such as liver cirrhosis, pulmonary fibrosis, chronic kidney disease,
and cardiovascular disease, are responsible for approximately 45% of deaths in industrialised nations, showing that fibrosis is a major global health problem and should be
Int. J. Mol. Sci. 2023, 24, 10237. https://doi.org/10.3390/ijms241210237
https://www.mdpi.com/journal/ijms
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better understood [1,2]. Fibrosis has been associated with excessive tissue deposition of
the extracellular matrix (ECM) due to the proliferation and activation of fibroblasts and
myofibroblasts that result in exacerbated growth of the injured tissue, forming hard scars
that destroy the normal architecture of the affected organ [3–5].
A wide variety of stimuli from different etiological origins, such as autoimmune reactions, allergic responses, tissue damage, and persistent infections, including parasitic
diseases, can trigger fibrotic processes [3–5]. One of the main causes of disease in schistosomiasis, caused by infections with Schistosoma spp., is fibrosis, and the liver is the main
affected organ in this case [6–9].
It is estimated that more than 250 million individuals are infected by Schistosoma spp.,
and approximately 780 million people live in areas at risk of infection [10]. Among all the
known species causing human schistosomiasis, Schistosoma mansoni is the most prevalent
in the human population and is the only species transmitted in the Americas [11,12]. The
morbidity of schistosomiasis is related to parasite load, time elapsed since infection, rate
of reinfection, nutritional status, and comorbidities [13–15]. These different factors will
determine the type and intensity of the host’s immune response elicited during infection,
and a chronic, exacerbated type-2 response has been associated with the degree of fibrosis
in this disease [13,15].
The initial immune response to S. mansoni infection is predominantly a type-1 response,
induced by larval and adult worm antigens [16,17]. When oviposition begins, egg antigens
are responsible for a shift towards a type-2 immune response, thereby triggering a heterogeneous extracellular matrix deposition around the parasite egg, which confines the cytotoxic
effects of secreted antigens and restrains tissue damage. However, the type-2 response also
leads to tissue fibrosis and portal hypertension, which is associated with the severity of
chronic schistosomiasis [18–23]. In parallel, an immunoregulatory response is induced and
becomes predominant around the 11th week onwards in most infected individuals [24–26].
The induction of an immunomodulatory response reduces T-helper (Th)2 inflammation,
affecting granuloma size and formation, and controls morbidity [25,27,28].
As many parasite eggs are swept through the hepatic portal system and become
trapped in the liver parenchyma, the induction and modulation of the immune response in
this organ are essential for the outcome of schistosomiasis mansoni [29,30]. In this disease,
resident liver cells play a key role in granuloma formation, tissue remodelling, and fibrosis.
Activation of hepatic stellate cells (HSCs) and myofibroblast differentiation are necessary
for collagen deposition and fibrosis in liver tissues, participating in granuloma formation
during schistosomiasis [7,31]. HSC activation can be influenced by a multitude of signals
from other resident and infiltrating cells, as well as autocrine and paracrine signalling from
HSCs [7]. Profibrotic cytokines, including transforming growth factor (TGF)-β1, connective
tissue growth factor (CTGF), IL-13, and IL-33, which are stimulated by S. mansoni egg antigens during granuloma formation, can also activate HSCs, triggering their differentiation
into myofibroblasts, thereby contributing to Schistosoma-induced fibrosis [32].
Therefore, fibrosis is preceded by inflammation, and elements of the innate and
adaptive immune responses are fundamental for the induction and regulation of fibrotic
processes [2]. IL-33 is an alarmin released by resident liver cells, such as hepatocytes,
endothelial cells, and HSCs, when Schistosoma eggs injure the host’s tissues [33,34]. The
biological functions of IL-33 depend on the activation of its only known receptor, the
suppressor of tumorigenicity 2 receptor (ST2), which is expressed in several liver-resident
and -infiltrating cells [35–37]. IL33/ST2 binding on type-2 innate lymphoid cells (ILC2s)
and eosinophils activates IL-13 production [36,38,39]. This cytokine, in turn, binds to the
IL-13Rα2 receptor in HSCs and activates extracellular signal regulated kinases 1 and 2
(ERK1-ERK2), subsequently triggering activin receptor-like kinases (ALKs) to activate Smad
1 and Smad 2, resulting in CTGF expression in HSCs and inducing the expression of type
I and III collagen, and matrix metalloproteinases [7,38,40]. Moreover, recombinant IL-33
was capable of activating HSCs in vitro, leading to the expression of IL-6, TGF-β, α-SMA,
and collagen [32]. The IL33/ST2 activation pathway also stimulates the early production of
Int. J. Mol. Sci. 2023, 24, 10237
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the Th2 cytokines IL-13 and IL-5, which increase the expression of cell adhesion molecules
(CAMs) in endothelial cells and leukocytes, thereby promoting infiltration and activation
of CD4+ T cells, macrophages, and eosinophils, besides activating tissue-resident cells, such
as fibroblasts [10,23,41–44].
The production of IL-33 has been associated with cellular recruitment, activation,
and ECM deposition in several experimental models of fibrosis. Bleomycin-induced lung
fibrosis has demonstrated that IL-33 promotes macrophage differentiation and activation
towards an M2 phenotype, collaborating with fibrosis [45,46]. Liver fibrosis induced by
thioacetamide and CCl4 administration or Helicobacter hepaticus infection also showed
the essential role of the IL-33/ST2 signalling pathway in the pathological alterations [47].
However, the role of IL-33/ST2 signalling in fibrosis caused by schistosomiasis remains
poorly understood.
In experimental infections with S. japonicum, exogenous administration of IL-33 leads
to larger liver granulomas and a stronger Th2 response, whereas inhibition of the IL33/ST2 pathway hinders fibrosis, collagen deposition, and differentiation of HSCs into
myofibroblasts [36,43,48,49]. In S. mansoni infections, Vanella et al. [34] reported that the
disruption of IL-33/ST2 signalling does not alter granuloma size or fibrosis and only
hampers the expression of Th2 cytokines when blocked, along with IL-25 and thymic
stromal lymphopoietin (TSLP) signalling. On the other hand, results from a study by
Maggi et al. [50] indicate that ST2 knockout mice have impaired modulation of granulomas
in the chronic phase of the schistosomiasis mansoni, leading to larger granulomas with an
increased cellular infiltrate, resulting in higher mortality.
Therefore, the effects of the IL-33/ST2 pathway on hepatic fibrosis and HSC activation
and differentiation during S. mansoni infection require further elucidation. In the current
study, we provide data demonstrating that IL-33/ST2 signalling is an essential step for HSC
activation during experimental schistosomiasis, and disruptions in this pathway result in
inappropriate granuloma organisation with altered extracellular matrix composition.
2. Results
2.1. Systemic Immune Response
To assess the systemic immune response during S. mansoni infection, we quantified
serum levels of IL-12p70, IL-13, IL-33, TGF-β, CCL24, and IL-22 (Figure 1). The majority
of serum samples tested from both mouse strains had no detectable levels of IL-12p70
and IL-13. We also observed that uninfected ST2−/− mice have higher serum levels of
IL-33, CCL24, and TGF-β when compared with WT mice. When infected, IL-33 levels
remain similar between mouse strains throughout the course of infection (Figure 1A).
Moreover, as expected, the serum concentrations of the regulatory cytokine TGF-β increased
progressively, peaking in the 12th week post infection in both WT and ST2−/− mice
(Figure 1B). In contrast, the serum level of CCL24, a chemokine that attracts eosinophils, and
IL-22, a cytokine associated with tissue repair mechanisms, showed significantly different
levels in WT and ST2−/− infected mice. Serum levels of CCL24 were significantly higher
in ST2−/− mice compared with WT mice between the 8th and 10th weeks of S. mansoni
infection; however, in the chronic phase of schistosomiasis, the serum levels of CCL24
were similar in both mouse strains (Figure 1C). IL-22 levels remained similar between the
2 strains until the 10th week of infection, while in the 12th week, the levels of this cytokine
were significantly reduced in infected ST2−/− mice (Figure 1D).
2.2. Liver Immune Response
Consistent with our previous data [50], the number of S. mansoni eggs retained in the
liver of both WT and ST2−/− mouse strains was statistically similar throughout the infection
(Figure 2A). Unlike that observed in serum, IL-33 levels in the liver are significantly lower in
ST2−/− mice compared with WT mice in both stages of infection (Figure 2B). Furthermore,
we observed that egg retention induces the activation of a Th2 response even in the absence
of the ST2 receptor, as seen by the concentrations of IL-4 and IL-13 in the liver; however, this
Int. J. Mol. Sci. 2023, 24, 10237
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response is not maintained in the chronic phase of infection, when ST2−/− mice showed
significantly lower levels of these cytokines than the infected WT mice (Figure 2C,D).
Figure 1. Serum cytokines levels. Evaluation of IL-33 (A), TGF-β (B), CCL24 (C), and IL-22 (D) levels
estimated by enzyme-linked immunosorbent assay (ELISA) inβ serum samples from WT (blue squares)
and ST2−/− (black triangles) mice. Mice were subcutaneously infected with 50 S. mansoni cercariae
−−
and the serum was collected at weeks 4, 8, 10, and 12 post infection. Data are shown as kinetics
and are presented as mean ± standard error of the mean (SEM). Normality was determined by the
Kolmogorov–Smirnov test. Comparisons between normally distributed groups were carried out by
−/− mice
−−
Student’s t-test,‐and p-values
in the same
‐ were assigned. Differences
ff between WT and ST2
phase of infection are represented by their p-value.
The concentrations of IL-17 in the liver homogenate were significantly higher in
knockout mice compared with WT mice in the acute phase of infection, but the levels of this
cytokine decreased significantly
in ST2−/− mice compared with WT mice in the chronic
−−
phase (Figure 2E). Just as in serum, the levels of IL-22 in ST2−/− mice are significantly
lower
compared with WT mice during chronic schistosomiasis (Figure 2F).
−−
2.3. Histopathology
In order to analyse how the IL-33/ST2 pathway affects the formation
of hepatic gran−−
ulomas, we evaluated haematoxylin-eosin (HE) liver slides (Figure 3). Histopathological
analysis revealed the presence of adult parasites in some liver sections within the portal
vessels of both infected mouse strains. Transverse sections of the parasite revealed a thin
tegument and digestive tube filled with a brown granular pigment (Figure 3A). Many
−−
vessels were hyperaemic and showed marked perivascular inflammation with moderate
−−
infiltration of plasma cells, lymphocytes, neutrophils, and eosinophils, associated with
ductal proliferation (Figure 3A). In acute schistosomiasis, livers from both infected groups
(Figure 3B) were characterised by multiple regions showing loss of parenchyma, which had
approximately 40% of its area replaced by inflammatory cells associated with schistosome
eggs and disorganised fibroblasts. Most of the inflammatory areas contained, centrally,
a schistosome egg surrounded by numerous neutrophils, eosinophils, lymphocytes, and
macrophages (Figure 3C,D). Fibroblasts intermixed with these inflammatory cells with
minimal to moderate quantities of collagen fibres formed additional layers in the outer
Int. J. Mol. Sci. 2023, 24, 10237
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zone of these areas and were generally more intense in liver granulomas from WT mice
(Figure 3C) than in ST2−/− (Figure 3D). Infiltration of multinucleated giant cells was less
frequent, and there were some pre-granulomatous areas. The remaining parenchyma
among the well-circumscribed lesion areas had a preserved tissue architecture; however,
scattered neutrophils and eosinophils were also seen between the cords of hepatocytes
or some foci of lesion. These focal lesions were characterised by necrotic foci associated
with fibrin, haemorrhage, moderate eosinophil infiltration, and macrophages containing
cytoplasmic hemosiderin.
Figure 2. Parasite load and immunological parameters in the liver. Quantification of eggs retained in
the liver of WT (blue squares) and
ST2−/− (black triangles) mice at 8 (acute) or 12 (chronic) weeks
−−
after infection with 50 cercariae (A). Quantification of Th2, Th17, and regulatory cytokines in liver
−−
homogenates from WT and ST2−/− BALB/c mice during acute and chronic infections. Mice were
subcutaneously infected with 50 S. mansoni cercariae and euthanised at weeks 8 (acute phase) or
12 post infection (chronic phase). Data compile results from two independent experiments and are
presented as dot plots and mean ± SEM, each point representing an animal, of the IL-33 (B), IL-4
(C), IL-13 (D), IL-17 (E), and IL-22 (F) concentrations estimated by sandwich ELISA. Normality was
determined by the Kolmogorov–Smirnov
test. Comparisons‐ between normally distributed groups
−−
ff
were carried out by two-way
ANOVA followed by Bonferroni’s post-test (A–C), while the Kruskal–
ff
Wallis test (D–F) was performed for non-parametric distributions, and the p-value were assigned.
Differences between WT and ST2−/− mice in the same phase of infection are represented by their
p-value. * represents significant differences between chronically infected mice in comparison with
mice of the same strain infected in the acute phase (* p < 0.05; ** p < 0.01; **** p < 0.001).
Int. J. Mol. Sci. 2023, 24, 10237
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Figure 3. Histopathology of hepatic granulomas of WT and ST2−− /− − mice infected with S. mansoni.
Photomicrograph of a HE-stained liver section with sections of adult worms within the portal vessel
of an infected ST2−−−/− mouse (A). Photomicrograph of a HE-stained liver section slide of granulomas
− − −/−
during acute phase from WT (C) and ST2
(B,D) mice and chronic schistosomiasis from WT (E)
−−
−
/
−
and ST2
(F). Scale bars = 100 µm (A,C–F) or 200 µm (B).
In chronic schistosomiasis,
the liver sections of infected mice from the WT and
ff
ST2−/− BALB/c groups were characterised by multifocal to coalescing areas with loss of
αparenchyma and replacement by well-formed granulomas associated or not with schistosome eggs. These areas replaced approximately 50–60% of the parenchyma. Inflammatory
cell infiltrate was comprised of macrophages, multinucleated giant cells, lymphocytes, neuαtrophils, and eosinophils in different proportions in the different stages of the granulomas
(Figure 3E,F).
Some recent necrotic
−−
α and acute inflammatory areas with viable eggs were
scattered throughout the parenchyma. The periportal areas had numerous plasma cells,
lymphocytes, and macrophages, and a mild increase in fibroplasia. Most of the liver granuα
lomas from infected WT mice showed lower cellular infiltration and had organised layers
of fibroblasts and eosinophilic fibres, concentrically arranged and surrounding the central
area (Figure 3E), while liver granulomas from infected ST2−/− mice still presented intense
cellular infiltration and more disorganised fibre deposition (Figure 3F). In some granulomas, the eggs were disrupted and partially or completely mineralised, with less frequent
Int. J. Mol. Sci. 2023, 24, 10237
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eosinophils. Pigmented (hemosiderin-containing) macrophages were comparatively higher
in these granulomas, although some were observed between the cords of hepatocytes.
In summary, the histopathological evaluation revealed differences in inflammation
and fibre deposition in liver granulomas between infected WT and ST2−/− BALB/c mice.
Thus, the livers from these mouse strains were further evaluated to reveal the pattern of
collagen deposition.
2.4. Hepatic Stellate Cell Differentiation
As the differentiation of HSCs into myofibroblasts is characterised by the expression
of α-SMA [7,51,52], the expression of this myofibroblast marker was evaluated in the livers
of the experimental groups. Immunohistochemical (IHC) analysis revealed that many cells
that composed Schistosoma-induced granulomas in the livers of infected WT mice expressed
α-SMA, especially in the chronic phase (Figure 4A). In contrast, liver granulomas from
infected ST2−/− mice showed very few α-SMA-expressing cells (Figure 4B). Morphometric
analysis of the brown-stained areas in hepatic granulomas of chronically infected mice
from both mouse strains confirmed a significantly lower expression of α-SMA+ cells in
granulomas formed in the absence of the activation of the IL-33/ST2 pathway (Figure 4C).
Figure 4. Analysis of myofibroblast activation in S. mansoni-induced hepatic granulomas. Photomicrographs of IHC-stained liver sections slides of granulomas from WT (A) and ST2−/− (B) mice after
−−
incubation with anti-α-SMA antibody in chronic schistosomiasis. Scale bars = 50 µm. Morphometric
α
analysis
showing the percentage of the area marked with α-SMA per field in hepatic granulomas
α chart bars show the mean ± SEM of three mice per
during the chronic phase of infection (C). The
group. Normality was determined by the Kolmogorov–Smirnov test. Comparisons between the
groups were carried out by the Mann–Whitney U test, and the p-value was assigned. Differences
‐
ff
between WT and ST2−/− mice in the same phase of infection are represented by their p-value.
−−
2.5. Collagen Deposition and Expression of Pro-Fibrotic Genes in Livers of S. mansoni-Infected Mice
−−
−−
‐
‐
To evaluate collagen production
and deposition in the hepatic granulomas
of WT and
ST2−/− mice, hydroxyproline levels were measured. The hydroxyproline concentration
in the liver increased significantly during the chronic phase of infection in both WT and
ST2−/− mice; however, there was no difference between the strains (Figure 5A). Histopathological analysis of the liver parenchyma stained with Masson’s trichrome, a stain specific for
collagen deposition, revealed that, although the hepatic hydroxyproline content in infected
ff similar, the pattern of collagen deposition in the granulomas
mice of both strains was
showed marked differences between groups, especially in the chronic phase of S. mansoni
infection (Figure 5B,C). In the chronic phase of schistosomiasis, the collagen deposition in
the liver granulomas of infectedttWT mice was compact and organised in well-delimited
fibres around
the eggs (Figure 5B), while in infected ST2−/− mice, the collagen was loosely
ff
and widely deposited around the parasite eggs with no well-defined fibre organisation
(Figure 5C).
−−
−−
−−
−−
Int. J. Mol. Sci. 2023, 24, 10237
ff
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Figure 5. Evaluation of collagen induction and deposition in the liver of WT and ST2−/− mice infected
−
with S. mansoni. Quantification of hydroxyproline concentrations in the livers of− WT
and ST2−/− mice
after experimental infection with S. mansoni (A). Photomicrographs of Masson’s Trichrome-stained
−−
hepatic granulomas of WT (B) or ST2−/− (C) mice chronically infected (14 weeks) with S. mansoni.
Scale bars = 50 µm. Relative quantification of mRNA expression of Col I (D), Col III (E), and Col
−−
VI (F) in the livers of WT and ST2−/− mice. RT-PCR was performed by extracting mRNA from
the liver homogenate from uninfected WT and ST2−/− mice or mice at weeks 8 (acute infection)
−−
and 12 (chronic
infection) of S. mansoni infection. Chart bars show mean ± SEM of 12 mice per
− group (D–F). Normality was determined by the Kolmogorov–Smirnov
group (A) and 5 mice −per
test. Comparisons between groups were performed by two-way ANOVA followed by Bonferroni’s
post-test, and p-values were assigned. Differences between WT and ST2−/− mice in the same phase
of infection are represented by their p-value. # represents significant differences between infected
mice and their uninfected controls; asterisk represents significant differences between chronically
−−
infected mice compared with mice
of the same strain in the acute phase of infection (# p < 0.05; ** or
## p < 0.01; #### p < 0.0001).
ff
ff
To assess the role of the activation of the IL-33/ST2 pathway on the expression of
genes associated with liver fibrosis in infected ST2−/− mice, qPCR analysis of collagen
genes was performed (Figure 5). Regarding mRNA expression of Col I (Figure 5D), the
S. mansoni infection induced a significant increase in Col I expression in WT and ST2−/−
mice, with no significant differences between mouse strains. As for Col III and Col VI, the
infection induced a significant increase in the mRNA expression of both genes in WT mice,
while in infected ST2−/− mice, the expressions of Col III (Figure 5D) and Col VI (Figure 5E)
were statistically similar to that of uninfected animals, both in the acute and chronic phases
of schistosomiasis. Thus, the mRNA expression of Col III and Col VI in the livers of infected
ST2−/− mice was significantly lower than in infected WT mice (Figure 5E,F).
The reduction of type III collagen fibres in liver granulomas of infected ST2−/− mice
was confirmed by Picrosirius staining. Although red-stained collagen fibres were observed
in the granulomatous reactions around parasite eggs trapped in the livers of both mouse
strains in chronic schistosomiasis (Figure 6A,C), the analysis of stained liver sections
under polarised light confirmed that granulomas from WT mice (Figure 6B) presented
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red and green collagen fibres, which represent type I and III collagens, respectively, with
overlapping type I and III fibres shown in yellow. In contrast, collagen fibres in liver
granulomas from infected ST2−/− mice were almost all red under polarised light, indicating
a clear predominance of type I collagen (Figure 6D).
Figure 6. Collagen fibre deposition on Picrosirius Red staining in hepatic granulomas of WT and
/− mice chronically infected with S. mansoni. Photomicrographs of liver sections from infected
ST2− −
−
WT and ST2−−− /− mice stained with Picrosirius Red. Scale bar = 100 µm. Hepatic granuloma of
WT mouse in chronic phase under optical microscopy (A) and under polarised light (B). Hepatic
−/− mouse in chronic phase under optical microscopy (C) and under polarised light
−
granuloma of− ST2
(D). The green arrow indicates the presence of collagen III (greenish colour), and the yellow arrow
indicates the presence of collagens I and III (yellowish colour) (B), while the presence of collagen I
(reddish colour) is indicated by a red arrow (D).
As indicated by Gomori’s ammoniacal silver staining of livers from S. mansoni-infected
mice, the reduction of collagen III and VI deposition in hepatic granulomas formed in the
absence of the activation of the IL-33/ST2 pathway was accompanied by modifications in
the arrangement of fibres (Figure 7). As shown in Figure 7B and confirmed by morphometric
analysis (Figure 7C), liver granulomas from chronically infected ST2−/− mice showed a
significant decrease in the formation of reticular fibres (Figure 7B,C). In contrast, WT mice
developed granulomas with well-organised reticular fibres around the eggs, which can be
seen as the black-stained regions on the periphery of the egg (Figure 7A).
−−
−−
−−
‐
ff
−−
−−
−−
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Figure 7. Reticular fibres of collagen in hepatic granulomas from S. mansoni-infected
WT and
−−
−/− (B) mice
ST2−/− mice. Representative photomicrographs of liver sections from
WT
(A)
and
ST2
−−
chronically infected with S. mansoni stained with Gomori’s Ammoniacal Silver. Scale bar = 50 µm.
Morphometric analysis of reticular fibres in liver sections from WT (blue squares)
and ST2−/− (black
−−
triangles) mice during the acute and chronic phases of infection (C). Data are represented as a dot
plot and mean ± SEM, each point representing the average of 20 granulomas per mice, with 4–8 mice
per group. Normality was determined by the Kolmogorov–Smirnov test. Comparisons between
groups were performed by Student’s t-test, and p-values were assigned. Differences between
WT and
−−
‐
ff
−
/
−
ST2
mice in the same phase of infection are represented by their p-value.
3. Discussion
Parasitic diseases such as schistosomiasis are an important cause of liver fibrosis. Liver
lesions caused by the S. mansoni infection are initiated by the retention of eggs in the hepatic
capillaries, which activate elements of the innate and adaptive immune responses and lead
to the formation of granulomas, a process that often results in liver fibrosis, associated with
the severity of chronic schistosomiasis [7,24,53–55]. In the current study, we evaluated the
role of the IL-33/ST2 activation pathway in the development of liver fibrosis caused by the
experimental S. mansoni infection. Our data revealed a significant reduction of pro-fibrotic
cytokines, such as IL-13 and IL-17, and the tissue-repairing IL-22 in the livers of chronically
infected ST2−/− mice. Moreover, infected ST2−/− mice showed lower activation of HSCs
and, consequently, lower differentiation of these cells into myofibroblasts. The decreased
myofibroblast differentiation was accompanied by a reduced expression of type III and
type VI collagen and lower formation of reticular collagen fibres in liver granulomas of
infected ST2−/− mice, indicating that the IL-33/ST2 activation pathway participates in liverrepairing mechanisms and the appropriate organisation of collagen around parasite eggs.
Interventions in particular immune pathways, such as IL-33/ST2 signalling, may
impact pathology as long as they can interfere with broader immune-mediated mechanisms.
Vannella et al. [34] have shown that the ablation of the IL-33/ST2 pathway during S. mansoniinduced inflammation may only significantly alter type-2 responses in the lung and liver in
conjunction with the blockage of TSLP and IL-25. In addition, we observed in a previous
work [50] that abrogation of the IL-33/ST2 pathway in experimental S. mansoni infections
does not affect type-2 response, but is sufficient for inducing changes in global inflammation
on its own, compromising immunoregulatory processes in chronic schistosomiasis.
Confirming our published results [50], the current data also demonstrated that, during
experimental infection with S. mansoni, there is an activation of Th2 and Th17 responses,
regardless of the absence of the IL-33 receptor; however, the IL-22 levels decreased systemically and locally. IL-22 is a cytokine homologous to IL-10, as their receptors share the
IL-10Rb chain. Yet, unlike IL-10, which acts on immune cells, such as macrophages and
T cells, IL-22 performs its function in tissue cells, such as hepatocytes [56,57]. During inflammatory processes, IL-22 directly protects the host tissue from destructive damage from
the exacerbated immune response; in the liver, this cytokine promotes repair and tissue
regeneration by stimulating the proliferation and survival of hepatocytes [57–59]. Aside
from the possible role in tissue repairing, IL-22 and IL-10 may also play an important role in
the modulation of liver fibrosis, decreasing HSC activation and tissue inflammation [59–61].
Int. J. Mol. Sci. 2023, 24, 10237
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Interestingly, as demonstrated in our previous articles [50], ST2−/− mice also showed a
reduction in IL-10 levels from the acute to the chronic phase of the infection, which may
also be associated with the exacerbated inflammatory response developed by this strain.
HSCs are resident cells in the hepatic parenchyma, and the transdifferentiation of these
cells from their quiescent state to myofibroblasts results in pro-fibrotic and proliferative
activity, which plays a central role in the pathogenesis of liver fibrosis induced by different
harmful or inflammatory agents [62–64]. Experimental evidence indicates that activated
HSCs are also a major source of collagen deposition and are essential for liver fibrosis and
extracellular matrix remodelling in schistosomiasis [7,62]. However, the role of IL-33 in the
activation of HSCs and liver fibrosis induced by the deposition of Schistosoma spp. eggs
remain poorly understood [7,38,65]. Our data revealed that S. mansoni infection in ST2−/−
mice resulted in lower α-SMA expression in liver granuloma cells compared with infected
WT mice. Since α-SMA expression is a marker of myofibroblast differentiation [7,51], this
indicates that the IL-33/ST2 activation pathway is essential for the differentiation and
activation of HSC-derived myofibroblasts in the livers of S. mansoni-infected mice.
The weak activation of HSCs in ST2−/− mice, shown in this study, also corroborated
the findings of Tan et al. [32] in an experimental model of bile duct ligation (BDL) fibrosis. HSCs isolated from ST2−/− mice, even when stimulated with high concentrations
(100 ng/mL) of recombinant IL-33, could not be activated, nor could they activate the c-Jun
N-terminal kinase (JNK)/ERK/p38 transcription factors of the mitogen-activated protein
kinase (MAPK) pathway, which are associated with the functionality of the IL-33/ST2
pathway, thereby indicating that the biological effect of IL-33 is dependent on the activation
of the MAPK pathway and ST2 signalling in HSCs. By inhibiting the JNK/ERK/p38-MAPK
pathway in HSCs isolated from C57BL/6 mice, a drastic decrease in collagen production
was observed, indicating that IL-33/ST2 signalling through this pathway is essential for
the activation and functionality of HSCs.
Furthermore, a previous study from our research group using the same experimental
model showed that the concentration of IL-13, which is one of the major factors involved
in the activation of HSCs and fibrosis, was also lower in the liver homogenate of ST2−/−
mice, while systemic and local TGF-β levels remain similar between both strains infected
with S. mansoni [38,50]. Kaviratne et al. [66] demonstrated that IL-13−/− mice infected with
S. mansoni presented an almost complete abrogation of the fibrosis process, despite the
continuous and unaltered production of TGF-β. The same study also demonstrated that,
by inhibiting a part of the TGF-β signalling cascade during experimental schistosomiasis
mansoni, there was no change in the development of fibrosis or the production of IL13, suggesting that the process of liver fibrosis induced by S. mansoni infections, while
dependent on IL-13, is TGF-β-independent. As demonstrated in other models of liver
fibrosis, IL-13 can be activated via the IL-33 pathway, implying that the liver fibrosis
mechanisms during schistosomiasis are mainly driven by a balance between IL-33 and
IL-13 instead of TGF-β [7]. Our data further support that the sustained TGF-β levels are not
sufficient for the induction of proper fibrosis in S. mansoni infections when the IL-33/ST2
pathway is not active. However, the lower levels of IL-13 during the chronic phase may
have prevented appropriate extracellular matrix deposition, thus affecting granuloma
formation in the absence of IL-33/ST2 signalling.
Other than that, a Th17 response has also been associated with granuloma formation
and fibrosis [67,68], whose activation is related to TGF-β and IL-6 [69,70]. Our data show
that the levels of IL-17 and IL-22, cytokines produced by Th17 cells, were affected by the
lack of IL-33/ST2 signalling. The increased IL-17 concentrations during acute infection may
have contributed to the observed unchecked periovular inflammation since this cytokine
has been associated with increased granuloma size and pathogenesis in schistosomiasis [68,71]. Despite the lower IL-17 levels in chronic infection, they are accompanied by
reduced IL-22 levels, both systemically and locally. The concomitant reduced IL-22 levels
could have not been sufficient to compensate for the expectedly decreased IL-17-driven liver
damage. Therefore, Th17-mediated pathogenesis could still be responsible for the main-
Int. J. Mol. Sci. 2023, 24, 10237
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tained granuloma size during the chronic phase. Supporting our findings, Nady et al. [72]
observed that the in vitro formation of granulomas induced by S. mansoni soluble egg
antigen (SEA) significantly decreased in the presence of IL-22 and IL-17 together, while
in the presence of IL-17 alone, there is granuloma growth due to the inflammatory role of
this cytokine.
Despite the compromised differentiation of HSC-derived myofibroblasts during S. mansoni infection in ST2−/− mice, the hydroxyproline content in the livers of these animals,
an indirect measure of tissue collagen deposition, was similar to that of infected WT mice.
Corroborating our data, Vannella et al. [34] also observed high and similar levels of hydroxyproline in the livers of IL-33−/− mice infected with S. mansoni. Although our data showed
that the tissue content of hydroxyproline was similar between the two strains of mice
analysed, the differences in the deposition of collagen in the granulomas between WT and
ST2−/− animals, as portrayed by Masson’s trichrome staining, were markedly significant.
ST2-deficient mice deposited collagen in a completely disorganised and exudative manner.
Consistent with the increase in hydroxyproline in infected mice, there was an increase in
Col I mRNA expression in the livers of infected WT and ST2−/− BALB/c mice, whereas
collagens III and VI increased only in the WT mice. Type I and III collagen production
and deposition are commonly found in schistosomiasis granulomas, with collagen I being
prominent during the chronic phase of schistosomiasis [6,73,74].
Our data showed that Col I expression was maintained in the absence of the IL33/ST2 pathway, but the lack of the ST2 receptor significantly reduced Col III and Col VI
expression in the acute phase of schistosomiasis, which was confirmed by histological
analysis with Picrosirius staining under polarised light. The induction of Col I expression
in infected ST2−/− animals may be related to the initial increase in the liver levels of IL-17,
as this cytokine is described as a potent inducer of type I collagen production [64,75,76].
Meanwhile, the reduction in type III collagen levels is in accordance with data found in the
literature for the human and murine tendinopathy models, in which it was possible to relate
IL-33 with type III collagen synthesis, showing that this cytokine plays a key role in the
transition of type III collagen synthesis through the regulation of miRNA29a [77]. The lower
production of type III and VI collagens in the absence of ST2 receptor activation resulted in a
reduced deposition and greater disorganisation of reticular fibres in these animals. Reticular
fibres are continuous collagen fibres composed of type I and III collagens in association with
other collagens, such as V [78], and this network of fibres helps to protect the injured tissue,
thereby contributing mainly to the containment of egg antigens [23,53]. As a result of this
disorganisation in the deposition of the extracellular matrix and the significant reduction in
the formation of reticular fibres in the Schistosoma-induced granulomas formed in ST2−/−
mice, these structures may not correctly confine the antigens released by S. mansoni eggs,
developing greater microscopic liver lesions and, consequently, higher mortality [50].
This difference in the production of collagen types was observed in another experimental model, as demonstrated by Li et al. [45] in a model of pulmonary fibrosis induction in
ST2−/− and WT C57BL/6 mice subjected to inoculation with bleomycin. In these animals,
although bleomycin stimulated type I collagen expression in both mouse strains, there was
a significant reduction in Col III expression in ST2−/− mice, which was associated with
early fibrosis repair. When WT mice received mIL-33 together with bleomycin, an increase
in inflammatory cells, such as alternatively activated macrophages (M2) and ILC2, was
observed, which increased the production of IL-13 and TGF-β, soluble collagen, and the
expression of Col III, consequently increasing the fibrosis score.
In addition to HSCs, alternatively activated macrophages play a special role in the
fibrosis process. Alternatively activated macrophages typically express low levels of
inflammatory cytokines and high levels of TGF-β, which promote collagen expression and
fibrosis [79]. Experimental evidence indicates that M2 macrophages play a key role in the
development of fibrosis and tissue repair in schistosomiasis [6,80,81]. Considering its role
as an alarmin with a type-2 immune profile, IL-33 seems to be a potential activator of M2
macrophages during innate and adaptive immune responses, as they strongly amplify
Int. J. Mol. Sci. 2023, 24, 10237
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the expression of arginase I and cytokines that contribute to fibrotic processes [82]. Some
authors have already reported this linkage of IL-33 with macrophages and the development
of pulmonary [45], renal [83], and liver fibrosis due to S. japonicum infection [48]. However,
there is still little clarification, and further studies are needed on the role of IL-33 in the
activation and differentiation of macrophages involved in liver fibrosis during S. mansoni
infection, which may be the key point for understanding the development of this process.
4. Methods
4.1. Mice
Six-to-eight-week-old female WT and ST2−/− BALB/c mice were used in the experiments. WT BALB/c mice were acquired from an established colony at the university mouse
facility. ST2−/− mice, deficient in the receptor that is activated by IL-33 binding [84], were
originally purchased from Jackson Immuno Research Laboratories and kindly provided by
Dr. João Santana (Faculdade de Medicina de Ribeirão Preto, USP, Brazil). Both strains were
maintained at the animal facility for infected animals of the Department of Parasitology
(ICB, UFMG, Brazil). Experimental mice were fed standard chow (Presence, Primor, Brazil)
and provided tap water ad libitum. The experimental procedures were approved by the
Ethics Committee on Animal Use (protocols 104/2007, 159/2012, and 368/2018) of the
Universidade Federal de Minas Gerais (UFMG, Belo Horizonte, Brazil).
4.2. Parasite and Infection
S. mansoni from the LE strain, originally isolated from a human patient in Belo Horizonte, Brazil, has been maintained by successive passages through Biomphalaria glabrata
snails and Mesocricetus auratus hamsters at the Laboratory of Schistosomiasis and Helminth
Immunology of the Department of Parasitology (ICB, UFMG, Brazil), and it was used for
the experimental infections performed in this study. S. mansoni cercariae were harvested
from infected B. glabrata snails, washed, counted, and subcutaneously injected into each
mouse (25 or 50 cercariae per mouse, as defined for each experiment), as previously described by Pellegrino and Macedo [85]. To confirm parasite infection and burden, the left
liver lobe of each infected mouse was digested in a 5% KOH solution, and the number of
recovered parasite eggs was estimated, as described previously [86].
4.3. Experimental Design
To evaluate the role of IL-33/ST2 activation on the liver pathology and fibrosis associated with S. mansoni infection, WT and ST2−/− BALB/c mice were infected with 25
or 50 cercariae and followed for up to 14 weeks. During this period, animals showing
severe clinical signs of schistosomiasis, such as accentuated weight loss and apathy, were
euthanised. At 0, 4, 8, 10, and 12 weeks after S. mansoni infection, 6 mice from each experimental group were immobilised, and blood was collected from the tail of each individual to
quantify cytokines and chemokines. At weeks 8 and 12 or 8 and 14 post infection (acute and
chronic phases of schistosomiasis, respectively), 6–10 mice from each experimental group
(WT and ST2−/− ) were euthanised by exsanguination via the brachial plexus under deep
anaesthesia by intraperitoneal injection of ketamine (80 mg/kg—Dopalen—Sespo Industry
and Commerce Ltda., Jacareí, Brazil) and xylazine (10 mg/kg—Kensol—Köing Laboratories
S/A, Avellaneda, Argentine). The left liver lobes of mice infected with 50 cercariae were
immediately removed and processed to quantify the gene expression of collagen types I, III,
and VI using real-time quantitative polymerase chain reaction (qPCR). Pathological evaluation was carried out in experiments with 25 cercariae, and the chronic phase was analysed
at 14 weeks. Due to the higher mortality rate of mice infected with a 50 cercariae inoculum,
they were analysed at the early chronic phase, i.e., 12 weeks post infection. The left liver
lobe was processed to estimate the number of parasite eggs retained in the parenchyma,
the median liver lobe was harvested to estimate the hydroxyproline content, and the right
liver lobe was promptly fixed and processed for histopathological analysis. Liver sec-
Int. J. Mol. Sci. 2023, 24, 10237
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tions were subjected to immunohistochemical procedures or were stained using either
haematoxylin-eosin, Masson’s trichrome, Picrosirius red, or Gomori’s ammoniacal silver.
4.4. Cytokine and Chemokine Assay
The concentrations of cytokines and chemokines were quantified in serum and liver
homogenate of WT and ST2−/− BALB/c mice by ELISA using commercially available kits
(Duoset, R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions.
In serum samples collected at weeks 0, 4, 8, 10, and 12 post infection, the concentrations
of IL-13, IL-12p70, IL-33, TGF-β, CCL24, and IL-22 were measured. To measure TGF-β,
each serum sample was activated with a HCl solution and diluted, as described by the
manufacturer. IL-4, IL-13, IL-33, IL-17, and IL-22 cytokine levels were quantified in liver
homogenate samples of all experimental groups, as previously described [50].
4.5. Quantification of Hydroxyproline
As an indirect determination of collagen deposition, the hydroxyproline content was
measured, as described previously [87]. A sample of the median liver lobe from each
experimental mouse was homogenised in 0.9% saline solution, frozen at −80 ◦ C, and
lyophilised (Liotop K105—Liobras, São Carlos, Brazil). The assay was carried out with
20 mg of the lyophilised product that was subjected to alkaline hydrolysis and treated with
chloramine T oxidising reagent, and the colorimetric reaction was assessed by the addition
of Ehrlich’s reagent [88]. The standard curve for the assay was prepared using serial
dilutions of known concentrations of hydroxyproline, and the absorbance was measured at
550 nm using a microplate reader (VersaMax, Molecular Devices, San Jose, CA, USA).
4.6. Histopathological, Immunohistochemical, and Morphometric Analyses of Liver
Histopathological evaluation of hepatic granuloma and collagen deposition was performed in liver sections from infected mice. Liver samples from each mouse were collected,
fixed in 10% buffered formalin, embedded in paraffin, and cut into 5 µm thick sections. The
liver sections were stained with haematoxylin-eosin to evaluate the overall inflammation
and tissue damage. Masson’s trichrome and Picrosirius red staining were performed to
illustrate extracellular collagen deposition around parasite eggs, and Gomori’s ammoniacal
silver staining to characterise the reticular fibre organisation [89–92]. Moreover, picrosiriusstained slides were examined under polarised light microscopy (Opticam, O500R, São
Paulo, Brazil) to characterise collagen composition; type I collagen appears as red fibres,
and type III collagen appears as green fibres under polarised light [89]. The HE and
picrosirius-stained images were captured with an Opticam LOPT14003 camera and analysed using OPTHD software (version x64, 4.10.17015.20200426). Liver sections stained with
Gomori’s silver from infected mice in both the acute and chronic phases of schistosomiasis
(6 mice/group) were used for morphometric analysis of reticular fibres. From each mouse,
20 microscopic fields containing granulomas were randomly selected, and the images were
taken with an Olympus Bx40 microscope with an attached Qcolor 3 camera (Olympus,
Tokyo, Japan). The area of the reticular fibres was quantified using the software package
Ks-400 (version V.3.0) [93].
Immunohistochemistry (IHC) was performed on slides with 5 µm liver sections of
mice from the different experimental groups to evaluate the expression of α-SMA. Each
section was deparaffinised, rehydrated, and subjected to antigen retrieval using a 0.01 M
sodium citrate solution (pH 6.0) by microwave heating at 80% power for 10 min. The
slides were then incubated with anti-α-SMA antibody (Abcam 32575, Cambridge, MA,
USA) at a dilution of 1:800 for 24 h at 4 ◦ C. After incubation with the primary antibody,
the slides were washed, and the presence of α-SMA in the liver parenchyma was revealed
using the EnVision+ Dual Link System-horseradish peroxidase (HRP) kit (Agilent Dako,
Santa Clara, CA, USA), in which the secondary antibody is associated with HRP-labelled
polymers, and peroxidase activity was revealed by the chromogen 3,3′ -diaminobenzidine
(DAB; Agilent Dako, Santa Clara, CA, USA). All slides were counterstained with Harris
Int. J. Mol. Sci. 2023, 24, 10237
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modified haematoxylin (Sigma-Aldrich, St. Louis, MO, USA) for 2 min and with bluing
reagent (Thermo Fisher Scientific, Waltham, MA, USA) for 1 min. Subsequently, the samples
were analysed under an optical microscope, and α-SMA was quantitatively estimated in
the livers of infected ST2−/− and WT mice using the ImageJ software (version 1.43). The
amount of α-SMA in granuloma cells was expressed as a percentage of the α-SMA-stained
area per field, analysing exudative granulomas containing viable miracidia from chronically
infected mice of both strains (3 mice/group), using an Olympus Bx41TF microscope coupled
to a DP12-camera 2 (Olympus Optical, Tokyo, Japan).
4.7. Quantification of mRNA Expression by RT-qPCR
The quantification of gene expression of collagen types I, III, and VI was performed in
liver samples using quantitative reverse transcription polymerase chain reaction (qPCR).
Liver fragments of mice from different experimental groups were collected, stored in
RNalater™ Stabilisation Solution (Invitrogen™, Waltham, MA, USA), and processed for
RNA extraction using a tissue homogeniser (Omni TH) and TRIzol kit (Invitrogen), according to the manufacturer’s instructions. After the quantification of RNA in a spectrophotometer (Epoch™ Microplate, Biotek, VT, USA), the samples were subjected to complete
digestion using a DNAse Turbo DNA-free kit (Ambion, Life Technologies, Thermo Fisher
Scientific, Waltham, MA, USA), followed by cDNA synthesis using a commercially available
High-Capacity cDNA reverse transcription kit (Ambion, Life Technologies). For quantification of gene expression, each PCR experiment was performed in triplicate in a total volume
of 10 µL, combining 5 µL of SYBR Green Master Mix (Ambion, Life Technologies), 0.3 µL of
each primer (forward and reverse) for the targeted gene, 2.4 µL of Milli-Q water, and 2.0 µL
of cDNA. The specific primers for the collagen type I, collagen type III, and collagen type
VI genes are listed in Table 1. The housekeeping gene GAPDH was used as an internal control. qPCR was performed in a StepOnePlus Real-Time PCR System (Applied Biosystems,
Waltham, MA, USA) using the following parameters: Taq polymerase was activated at
95 ◦ C for 10 min, followed by 40 cycles of amplification (95 ◦ C for 15 s for denaturation, and
60 ◦ C for 60 s for primer annealing and extension). Relative quantification of the mRNA
expression for each target gene was performed using the 2∆CT method [94]. Data were
analysed using StepOne 2.3 (Life Technologies).
Table 1. Primer sequences for GAPDH, Collagen type I, Collagen type III, and Collagen type VI used
in the RT-qPCR.
Sequence (5′ –3′ )
Gene
GAPDH
Forward
AGG TCG GTG TGA ACG GAT TTG
[95]
Reverse
TGT AGA CCA TGT AGT TGA GGT CA
Col I
Forward
ACT GGA CTG TCC CAA CCC C
[95]
Reverse
CTT AGT TTG GAC AGG ATC TGG
Col III
Forward
AAC CTG GTT TCT TCT CAC CCT TC
[94]
Reverse
ACT CAT AGG ACT GAC CAA GGT GG
Col VI
Forward
CGC CCT TCC CAC TGA CAA
[96]
Reverse
GCG TTC CCT TTA AGA CAG TTG AG
4.8. Statistical Analysis
Statistical analysis was performed, and graphs were generated using GraphPad Prism
8.0.2. Data were subjected to ROUT tests for the identification and removal of outliers, and
their normal distribution was verified using the Kolmogorov–Smirnov test. Comparisons
between the experimental groups at different time points of infection were carried out
by two-way ANOVA, followed by Bonferroni’s post-test, and comparisons between only
one phase of infection were carried out by Student’s t-test. For non-parametric datasets,
Int. J. Mol. Sci. 2023, 24, 10237
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comparisons between multiple groups were performed by the Kruskal–Wallis test, while
comparisons between only two groups were carried out by the Mann–Whitney U test. After
being assigned, p-values ≤ 0.05 were considered significant.
5. Conclusions
The severity of schistosomiasis has been associated with the intensity and extent of
tissue damage and fibrosis induced by parasite eggs trapped in the host’s tissues. This
study shows that, in S. mansoni-infected mice, the IL-33/ST2 activation pathway did not
significantly change the quantity of collagen deposited around the parasite eggs retained
in the host’s liver. However, activation of this pathway participates in the induction of
some pro-fibrotic cytokines, such as IL-13 and IL-17, and the tissue-repairing IL-22 in the
livers of chronically infected ST2−/− mice. Moreover, IL-33/ST2 signalling is essential
for the differentiation of HSCs into myofibroblasts and, consequently, for the production
of type III and VI collagen. This results in the proper organisation of collagen reticular
fibres within the granulomatous reaction, enabling the adequate development of hepatic
granulomas and antigen retention during schistosomiasis, protecting the liver parenchyma.
Thus, the current study provides evidence that supports the role of the IL-33/ST2 pathway
in liver-repairing mechanisms and the appropriate organisation of collagen around parasite
eggs, participating in S. mansoni-induced liver fibrosis and controlling disease severity.
Author Contributions: L.M. and D.A.N.-C. conceptualised and designed the study; L.M., G.M.A.C.
and I.C.R. performed animal experiments and data analysis; L.M. and W.P.A. conducted the q-PCR
procedure and data analysis; É.M.L.R. supervised and provided resources to carry out the q-PCR
experiments; T.A.P. performed the immunohistochemical analysis; A.C.J. and J.M.P.M. executed the
morphometric analysis; W.L.T. and R.E. supervised the histopathological analysis; L.M., G.M.A.C.,
R.E. and D.A.N.-C. wrote the manuscript. All authors have read and agreed to the published version
of the manuscript.
Funding: Financial support for this work was provided by the Fundação de Amparo à Pesquisa do
Estado de Minas Gerais (FAPEMIG-Brazil, grant #PPM-00500-15 and grant #APQ 01637-17). D.A.N.C., L.M. and G.M.A.C. were supported by a fellowship from CNPq/Brazil. J.M.P.M. was supported
by CAPES/Brazil.
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Ethics Committee on Animal Use of Universidade Federal de Minas Gerais (protocols code 104/2007,
159/2012, and 368/2018).
Informed Consent Statement: Not applicable.
Data Availability Statement: All data supporting our results is present in the paper.
Acknowledgments: Acknowledgement is due to José Carlos dos Reis for the technical support provided.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the study
design, data collection, and analysis, or decision to publish the manuscript.
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