Transcriptional Profiles of Cytokine/Chemokine Factors of Immune Cell-Homing to the Parasitic Lesions: A Comprehensive One-Year Course Study in the Liver of E. multilocularis-Infected Mice
et al. (2014) Transcriptional Profiles of Cytokine/Chemokine Factors of Immune Cell-Homing to the Parasitic
Lesions: A Comprehensive One-Year Course Study in the Liver of E. multilocularis-Infected Mice. PLoS ONE 9(3): e91638. doi:10.1371/journal.pone.0091638
Transcriptional Profiles of Cytokine/Chemokine Factors of Immune Cell-Homing to the Parasitic Lesions: A Comprehensive One-Year Course Study in the Liver of E. multilocularis -Infected Mice
Junhua Wang 0
Renyong Lin 0
Wenbao Zhang 0
Liang Li 0
Bruno Gottstein 0
Oleg Blagosklonov 0
Guodong Lu 0
Chuangshan Zhang 0
Xiaomei Lu 0
Dominique A. Vuitton 0
Hao Wen 0
Geoffrey N. Gobert, Queensland Institute of Medical Research, Australia
0 1 State Key Lab Incubation Base for Xinjiang Major Diseases Research and Xinjiang Key Laboratory of Echinococcosis, First Affiliated Hospital of Xinjiang Medical University , Urumqi, Xinjiang , China , 2 Department of Nuclear Medicine, University of Franche-Comte and Jean Minjoz University Hospital , Besanc on, Franche-Comte , France , 3 Institute of Parasitology, University of Bern , Bern , Switzerland , 4 WHO-Collaborating Centre for the Prevention and Treatment of Human Echinococcosis, University of Franche-Comte and University Hospital , Besanc on, Franche-Comte , France
Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL12a, IFN-c and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-b. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.
Funding: This work was supported by NSFC Grant Projects (81260452, 81260252), the Swiss National Science Foundation (31003A_141039/1), the Program for
Changjiang Scholars and Innovative Research Team in Universities (IRT1181), and Xinjiang Key-Lab Projects (SKLIB-XJMDR-2012-Y1). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Alveolar echinococcosis (AE) is a rare, but - if remaining
untreated or treated too late- severe and fatal zoonotic helminthic
disease, predominantly caused not only by the direct hepatic
damage which follows the continuous tumor-like proliferation of
the larval stage (metacestode) of Echinococcus multilocularis
(E.multilocularis), but also indirectly by the intense local granulomatous
immune response which surrounds the parasitic tissue .
Granuloma, extensive fibrosis, and necrosis are actually the
characteristic pathological findings in E. multilocularis infection.
The lesions, composed both of the multiple vesicle-forming
metacestode and of cells homing from lymphoid organs and
permanently settling around the metacestode, behave like a
slowgrowing liver cancer, progressively invading the liver, then the
neighboring tissues and also metastazing to other organs .
Pathological changes in AE are associated with an intense
infiltration by immune cells, i.e. macrophages of various functional
types, including the so-called epithelioid cells and giant cells,
typical of granulomas  and T lymphocytes. CD4+ T
lymphocytes are present from the early stage of parasite growth and CD8+
T lymphocytes are known to home to the periparasitic infiltrate
secondarily and to be associated with parasite tolerance and
severity of the disease [1,2,3,4]. Non-immune cells such as
fibroblasts and myofibroblasts which are crucial for the
development of fibrosis are also attracted by the hosts immune response
around the parasite.
It has been shown that E. multilocularis infection induced
numerous pathways of the immune response; the involvement of
individual cytokines has been rather extensively studied within the
past 2 decades both in humans and in experimental rodents . In
the immune-competent but susceptible host, E. multilocularis
induces skewed Th2-responses . In chronic AE, Th2-dominated
immunity is associated with increased susceptibility to disease,
while Th1 cell activation induces a rather protective immunity
which involves IFN-a  and IL-12  as initiating cytokines, and
IFN-c  and TNF-a [9,10] as effector cytokines. During the
course of E. multilocularis infection, as studied in mice, an initial
acute stage Th1 response gradually switches to an increasingly
dominating Th2 response; the thus mostly mixed Th1/Th2 profile
of the chronic stage is associated with the expression of
proinflammatory cytokines in the granuloma [11,12]. Th2 cytokines
down-modulate the Th1 response which nevertheless decreasingly
persists all along the infection until the late pre-mortem
immunesuppressed stage of AE . The metacestode actively achieves a
tolerance status through the induction of regulatory cytokines,
such as IL-10 and TGF-b . However, this bulk of information
has mostly been obtained from studies on peripheral blood
mononuclear cells (in humans), and on spleen and lymph node
cells in the experimental model [5,13,14]. In addition, nothing was
known until very recently about role of IL-17 and Th17 cells
[13,14] during E. multilocularis infection. Only two studies have
given some insight into chemokine [15,16] and IL-17 
involvement in E. multilocularis infection, respectively; and this
was done only in AE patients, and never in the infected liver tissue;
the actual involvement of IL-17 and chemokines in the lesions is
thus unknown. The time course of IL-17 expression is also
unknown since human AE is usually discovered late, i.e. years after
E. multilocularis infection of the patients, and findings in humans
reflect only the late chronic stage of infection. Studies in the
experimental mouse model are therefore necessary to dissect the
various stages of E. multilocularis infection regarding the hosts
In the present report, our objectives were to 1) give a
comprehensive appraisal of the various components, especially
cytokines and chemokines, involved in immune cell homing
around the E. multilocularis metacestode, at the various successive
stages of disease, i.e. early, middle and late stages as defined
previously [18,19], and 2) to study the parasite and the host
immune response in their usual context, the liver, in the
experimental mouse model of hepatic secondary infection.
Eighteen key-cytokines and -chemokines were measured both in
the lesion, including the periparasitic infiltrate, and in the
surrounding liver, close to the lesions, using qRT-PCR. To get a
more complete picture of the influence of the parasite-induced
hosts immune response on the hosts liver, a microarray technique
was also used to study the surrounding liver tissue.
Hepatic histopathology during E. multilocularis-infection
From day 2 to day 360 post-infection (p.i.) with E. multilocularis,
the hepatic parasitic lesions showed the various morphological
patterns specific to the different stages of murine AE, as described
in a previous study using the same experimental mice (data not
shown) [18,19]. According to previous reports on the course of E.
multilocularis secondary infection in experimental susceptible mice
[18,19], the 3 main stages were defined as follows: early stage,
from infection to day 60; middle stage from day 60 to day 180; and
late stage from day 180 to day 360.
Innate immunity and pro-inflammatory cytokines
In E. multilocularis parasitic lesions (i.e. including adjacent
infiltrates, as defined in the Materials and Methods section),
qRTPCR showed that IL-12a mRNA expression was 6.3-fold higher at
as early as day 2 p.i. than in control mice (Figure 1A). There was a
significant difference between E. multilocularis-infected mice and
control mice, at the early stage of infection, at time points of 2-,
8and 30-day p.i. (P,0.05). In the periparasitic liver tissue (i.e. liver
parenchyma close to the lesions, as defined in the Materials and
Methods section), IL-12a mRNA expression was also higher than
in control livers from day 8 to day 30 p.i.. There was a significant
difference at 30-days p.i. (P,0.05). Changes in IL-12a mRNA
expression with time are shown in Figure 1A.
In E. multilocularis lesions, qRT-PCR showed that TNF-a
mRNA expression was increased at the early stage of infection,
especially at days 2 and 8 p.i.; it remained high at 30 days p.i. but
decreased subsequently (Figure 1B). There was a significant
difference between E. multilocularis infected mice and control mice,
at the time points of 2-, 8- and 30-day p.i. (P,0.05). In the
periparasitic liver tissue, TNF-a mRNA expression did not change
from day 2 to day 360 (Figure 1B). In the lesions, there was an
increase in IL-1b mRNA expression all over the infection course,
from day 2 to day 360 p.i., with a peak at 60 days p.i.. IL-1b
mRNA expression was 2.5-fold higher at day 2 and 7.6-fold higher
at day 60 (Figure 1C), when compared to control mice. There was
a significant difference between E. multilocularis infected mice and
control mice, at the time points of 30-, 60-, 90-, 270- and 360-days
p.i. (P,0.05). In the liver tissue, IL-1b mRNA expression
increased later, from 2.9-fold at day 30 to 4.7-fold at day 90
(Figure 1C), and was at its maximum at the middle stage of
infection. There was a significant difference at the time points of
30-, 60- and 90-days p.i. (P,0.05). In the lesions, IL-6 mRNA
expression was markedly increased as early as 2 days; then it
relatively decreased at day 30 p.i., then re-increased very
significantly from day 90 p.i. (4.8-fold) (Figure 1D). There was a
significant difference between E. multilocularis infected mice and
control groups, at the time points of 2-, 60-, 90-, 180- and
360days p.i. (P,0.05). In the liver, IL-6 mRNA expression increased
at the very early stage of infection, 1.8-fold at day 2 and1.9-fold at
day 8 (Figure 1D); it returned back to normal at day 30, and
reincreased from day 60 to day 90, then a high level was maintained
until day 360.
Th1 cytokines and related chemokines
Th1 cytokines. In the lesions, an increase in IFN-c mRNA
expression was observed from day 2 to day 360 p.i., with a peak at
30 days p.i.. Except for an apparent decrease at day 8, IFN-c
mRNA-expression was especially increased at the early stage of
infection, from 3.6-fold at day 2 to 4.8-fold at day 30 (Figure 2A).
There was a significant difference between E. multilocularis infected
mice and control mice, at the time points of 2-, 30-, and 60-day
p.i., but also at the latest stage, 360- day p.i. (P,0.05). In the liver,
IFN-c mRNA expression was increased from 2.4-fold at day 2 to
3.1-fold at day 30 (Figure 2A), but became abrogated at the late
stage of infection, from 0.5- fold at day 90 to 0.4 at day 360,
compared to control mice. There was a significant difference at the
time point of 30-day p.i. (P,0.05).
Th1-related chemokines. Expression of CXCL9 mRNA
was observed from day 2 to day 360 p.i.. In the lesions, CXCL9
mRNA expression was increased from day 90 to day 360, with a
peak of 9.75-fold at day 180 (Figure 2B), compared to control
mice. There was a significant difference between E.
multilocularisinfected mice and control mice, at the time points of 2-, 8-, 90-,
180- and 270-days p.i., i.e. at the late stage of infection (P,0.05).
In the liver, CXCL9 mRNA expression was increased by 1.72-fold
at day 2 and 2.78-fold at day 8 (Figure 2B); it was decreased by
0.30- fold at day 30 and by 0.21-fold at day 60, then expression
reincreased by 3.5- fold at day 90 compared to control mice. There
was a significant difference at the time points of 8- and 90-day p.i.
(P,0.05). In the lesions of E. multilocularis-infected mice, CXCL10
mRNA expression was increased by 1.6-fold at day 2; then levels
progressively increased to a peak (7.8-fold the levels in control
mice) at day 90 p.i. (Figure 2C). There was a significant difference
between E. multilocularis infected mice and control mice, at the time
points of 30-, 60- and 90-days p.i. (P,0.05), i.e. at the middle stage
of infection. In the liver, CXCL10 mRNA expression was
increased at day 60 (2.1-fold) and at day 90 (2.2-fold)
(Figure 2C), and was lower both at the early stage and the late
stage of infection when compared to control mice. Expression of
the mRNA of CXCL12, a chemotactic factor for lymphocytes, was
observed from day 2 to day 360 p.i.. In the lesions, CXCL12
mRNA expression was markedly increased as early as day 2
postinfection, when it reached a peak (11.6-fold); it remained elevated
until day 60(Figure 2D). There was a significant difference
between E. multilocularis infected mice and control groups, at the
time points of 2-, 8- and 60-days p.i. (P,0.05). In the liver,
CXCL12 mRNA expression was increased early, from 1.1-fold at
day 2 to 2.1-fold at day 8 (Figure 2D), and was lower than that
observed in control mice at the late stage, from day 90 to day 360.
There was a significant difference at the time points of 8- and
270days p.i. (P,0.05).
Th2 cytokines and related chemokines
Th2 cytokines. In E. multilocularis lesions, IL-4 mRNA
expression followed a biphasic curve: it was increased early
(3.8fold at day 2), and was significantly different from that observed in
control mice at 2 and 8 days; but it relatively decreased at 30 p.i.; it
then re-increased and was still elevated at the late stage [4.2-fold at
day 360; significantly different from control mice (P,0.05).
(Figure 3A)]. In the liver, IL-4 mRNA expression was increased
compared to control mice [4.8-fold at day 8 and 3.2-fold at day 60,
significantly different from control mice (P,0.05) (Figure 3A)]. In
E. multilocularis lesions, IL-5 mRNA expression was present from
the early stage (2.3-fold at day 2); however (Figure 3B), there was a
peak of 13.6-fold at day 90, and a significant difference between E.
multilocularis infected mice and control mice, all over the middle
and late stages of infection, at the time points of 60-, 90-, 180- and
360-days p.i. (P,0.05). In the liver, IL-5 mRNA expression was
also markedly increased at the middle stage of infection: 3.5-fold at
day 60 and 6.54-fold at day 90 (Figure 3B). There was a significant
difference at the time points of 60- and 90-days p.i. (P,0.05).
Th2-related chemokines. In the lesions, mRNA expression
of CCL8, chemotactic for and activator of various immune cell
types, including mast cells, eosinophils and basophils, monocytes,
T cells, and NK cells , was increased from day 2 to day 360
p.i., with a peak at day 90 (Figure 3C). There was a significant
difference between E. multilocularis infected mice and control mice,
at the very early and at the middle stage of infection, at the time
points of 8- and 90-days p.i. (P,0.05). In the liver, there was no
difference in CCL8 mRNA expression from day 2 to day 360
(Figure 3C) between infected and control mice. In the lesions,
mRNA expression of CCL12, another Th2-related chemokine,
which attracts eosinophils, monocytes and lymphocytes ,
increased early, from 2.0-fold at day 2 to 6.6-fold at day 8 p.i.
when it became significantly different from control mice
(Figure 3D); levels were also elevated at day 90 p.i. (3.5-fold; also
significantly different from control mice). In the liver, CCL12
mRNA expression did not change from day 2 to day 360
(Figure 3D), compared to control mice. mRNA expression of
CCL17, which induces T-cell chemotaxis and elicits its effects by
interacting with the chemokine receptor CCR4, was observed in
the lesions (1.7- fold increase at day 2 and 2.0-fold at day 180 p.i.),
(Figure 3E). There was a significant difference between E.
multilocularis infected mice and control groups, at the time points
of 8-, 60- and 90-days p.i., when its expression peaked at 3.7 fold
(P,0.05). A slight decrease in CCL17 mRNA expression was
observed at day 30 p.i., concomitant to the slight decrease also
observed for the Th2-related cytokines IL-4 and IL-5. In the liver,
CCL17 mRNA expression was higher than in control mice from
day 2 to day 180 (Figure 3E). There was a significant difference at
90-days p.i. between infected and control mice (P,0.05).
IL-17 and its isotypes. In the periparasitic infiltrate area,
IL17, disclosed by immunostaining (Figure 4A), was observed in
most lymphocytes and macrophages in the periparasitic infiltrate,
as well as in fibroblasts, and endothelial cells in hepatic sinusoids,
especially around the granulomas, and in infiltrating immune cells
of portal spaces, from day 8 to day 360 p.i.. IL-17 positive scores
ranged from 0.13 to 4.80 and reached the peak point at day 90p.i.
(Figure 4B). In the liver close to the parasite lesions, moderate
IL17 expression was observed; there was a significant difference
between AE-infected and sham-injected mice at day-8, -30, -90,
270 and 360p.i..
In E. multilocularis lesions, IL-17A mRNA expression was
increased at the very early stage of infection, by 6.9-fold at day
2 and by 9.6-fold at day 8 p.i. (Figure 4C), and decreased at the
late stage, from day 180 to day 360 p.i.. There was a significant
difference between E. multilocularis infected mice and control
groups, at the time points of 2-, 8- and 90-days p.i. (P,0.05). In
the liver, IL-17A mRNA expression was also increased at the very
early stage: 6.7-fold at day 8; at this time point, the difference was
significant (Figure 4C) (P,0.05). In the lesion, IL-17F mRNA
expression was present all over the infection course, from day 2 to
day 360 p.i. (Figure 4D), with a peak of 5.63-fold at day 8
compared to control mice. There was a significant difference
between E. multilocularis infected and control mice, at the time
points of 8- and 60-days p.i. (P,0.05). At the late stage, despite an
apparent increase, compared to control mice, the difference was
not significant. In the liver, IL-17F mRNA expression did not
change significantly from day 2 to day 360 (Figure 4D).
Treg-related nuclear transcriptional factor and cytokines
Treg related nuclear transcriptional factor (Foxp3). In
E. multilocularis lesions, Foxp3 mRNA expression was increased by
2.4-fold at day 2 and by 3.0-fold at day 8 p.i. (Figure 5A); it then
decreased from day 30 to day 60 p.i., and re-increased, from
1.9fold at day 90 to 2.3-fold at day 360 p.i., with a peak of 3.1-fold at
day 180, at the late stage of infection (Figure 5A), thus following a
biphasic curve in the course of infection. There was a significant
difference between E. multilocularis infected mice and control mice,
at the time points of 2-, 8-, 180- and 360-days p.i. (P,0.05). In the
liver, there was no significant change in Foxp3 mRNA expression
Treg-related cytokines. In E. multilocularis lesions, TGF-b1
mRNA expression also followed a biphasic curve, with a decrease
at days 30 and 60 p.i.; it was increased by 3.6-fold at day 2 and
3.2-fold at day 270 p.i. (Figure 5B) with a peak of 5.7- fold at day
180 (Figure 5B). There was a significant difference between E.
multilocularis infected mice and control mice, at the early and late
stages of infection, at time points of 2-, 8-, 90-, 180-, 270- and
360day p.i. (P,0.05). In the liver, TGF-b1 mRNA expression was also
increased from day 8 to day 360 p.i., with a peak at day 180 p.i..
Conversely to the expression of TGF-b1 mRNA in the lesions, in
the liver, TGF-b1 mRNA was significantly elevated at the middle
and late stages, at the time points of 90-, 180- and 270-days p.i.
(P,0.05). In E. multilocularis lesions, IL-10 mRNA expression was
also biphasic, with a significant increase at the early and late stages
of infection, but not at its middle stage (Figure 5C). There was a
significant difference between E. multilocularis infected mice and
control mice, at the time point of 8-day, then at 180-, 270- and
360-days p.i. (P,0.05). In the liver, IL-10 mRNA expression did
not change from day 2 to day 360 (Figure 5C) compared to control
Immune response and inflammation gene expression in
the liver of E. multilocularis infected mice
To further give a comprehensive picture of the immune
response-related changes in the adjacent liver during E.
multilocularis infection, and especially detect hyper-expression of the
genes of cytokine/chemokine receptors, cDNA microarray
technology was used. The individual genes associated with the gene
ontology biological process immune response, and pathogen
response assessed at different time periods of infection, i.e. 30, 60,
90, 180 days p.i., are presented in Table 1. We used Gene
Ontology (GO; www.geneontology.org) analysis which clusters the
genes associated with immune response/defense (n = 59) into
functional subgroups including macrophages, APCs, chemokines
and chemokine receptors, lymphocytes, B-cells and eosinophils.
More precisely, at 30 days p.i., several biological processes
relating to an active infection, as defined by GO cluster
classification, were involved, including genes mostly associated
with the response to external stimuli, response to wounding,
immune response, response to stress, chemokine activity, defense
response, MHC-related functions and inflammatory response.
While several chemokine genes were found activated in the liver of
AE mice by qRT-PCR, microarray analysis did not show any
upregulation of cytokine genes. Among genes of cytokine receptors,
only those for IL-1 (IL-R1 like) and IL-7 (2.92 and 2.25 fold
respectively) were up-regulated at day 30 (Table 1). Among genes
encoding for chemokines, CCL5 (RANTES), a Th17-related
chemokine that up-regulates IL-12 and IFN-c, and is involved in
Th1 cell-migration , was up-regulated 2.35-fold at day 30.
Th2-related CCL8, CCL12 and CCL17 were up-regulated
29.58fold, 5.64-fold and 3.36-fold at day 30, respectively. Among genes
related to macrophage function, MGL1 and MGL2 (C-type
macrophage galactose-type lectins) were up-regulated 2.56- and
4.64-fold respectively, compared to control mice.
At 60 days p.i., genes involved in the response to stress, response
to external stimulus and response to biotic stimuli were added.
There were few changes in the immune response gene expression,
except for MPA2L (macrophage activation 2-like), which was
down-regulated 2.33-fold and C4b (Complement component 4B),
which was up-regulated 3.09-fold, respectively.
At 90 days p.i., among genes encoding for cytokine receptors,
IL-13 Ra1 was up-regulated 2.39-fold (Table 1). Among the
interferon-activated genes, Ifi202b, Ifi203 and Ifi204 were
upregulated 2.88-, 2.13-, and 2.47-fold, respectively. Among genes
encoding for macrophage functions, MSR1 (macrophage type-I
class-A scavenger receptors) and MPA2L (macrophage activation
2-like) were up-regulated 2.11- and 3.99-fold, respectively, when
compared to control mice.
At 180 days p.i., hyper-expression of genes of the inflammatory
response, response to stress, and response to external stimuli was
maintained, and genes of antigen processing and presentation,
complement activity and antigen processing via MHC class II
were also hyper-expressed. Among genes of cytokine receptors,
IL17R was up-regulated 2.90-fold (Table 1). Among genes encoding
for chemokines, CXCL9 was up-regulated 3.81-fold at day 180,
and CXCL12 was down-regulated 2.11-fold at day 180.
Correlations between mRNA levels of the various
cytokines over the course of infection
Spearman correlation coefficients indicated a significant positive
correlation between TGF-b1 mRNA expression in E. multilocularis
parasitic lesion, and that of Foxp3 (r = 0.719, P = 0.045), IL-10
(r = 0.761, P = 0.028) and CXCL9 (r = 0.946, P,0.01), but a
significant negative correlation with IFN-c (r = 20.743, P = 0.035)
(Table 2); it also showed a significant positive correlation between
Foxp3 expression in E. multilocularis parasitic lesion, as measured
by qRT-PCR, and IL-10 (r = 0.761, P = 0.028) and TNF-a
(r = 0.742, P = 0.035), but a significant negative correlation with
IL-1b (r = -0.754, P = 0.033) (Table 3). There was a significant
positive correlation between IL-17A expression in E. multilocularis
parasitic lesion, as measured by qRT-PCR, and CCL12
(r = 0.833, P = 0.011), CCL17 (r = 0.733, P = 0.039), IL-4
(r = 0.710, P = 0.049) and TNF-a (r = 0.804, P = 0.016) (Table 4);
there was also a significant positive correlation between IL-17F
mRNA expression in E. multilocularis parasitic lesion and CCL12
(r = 0.708, P = 0.049) and CCL17 (r = 0.749, P = 0.032)(Table 4).
TNF-a mRNA expression in E. multilocularis parasitic lesion was
also significantly correlated to IL-12a (r = 0.888, P = 0.033)
Despite the alleged causative involvement of the granulomatous
response in the clinical development of AE and its role in
functional imaging of the disease, since it is responsible for the
Fluorodeoxyglucose (FDG) uptake in Positron Emission
Tomography (PET) , a comprehensive picture of the cytokine/
chemokine response that occurs in situ, i.e. in the periparasitic
granuloma, had never been given. Chemokines and IL-17, which
are crucial for immune cell homing, have so far received little
attention in E. multilocularis infection. In the present longitudinal
study of experimental E. multilocularis intra-hepatic infection model,
we showed for the first time that 1) the mixed Th1/Th2/Treg
response and the tri-phasic course of cytokines, suggested by
previous studies on spleen cells from E. multilocularis-infected mice,
was also documented in the periparasitic infiltrate, but
nevertheless differed in some aspects, especially the marked and parallel
expression of IL-12a and TNF-a but also IL-4 at a very early stage
of the parasite/host interactions; 2) IL-17 was involved locally at
the beginning of the immune response and remained so all along
the course of infection, with a successive expression of different
macrophage activation 2 like
macrophage scavenger receptor 1
macrophage scavenger receptor 2
Pre B-cell leukemia transcription factor 1
Phospholipase A2, group VII (platelet-activating
factor acetylhydrolase, plasma)
Protein kinase C, theta
Serum amyloid A 1
Serum amyloid A 3
Serum amyloid A 4
Serine (or cysteine) peptidase inhibitor,
clade A, member 3K (Serpina3k)
Serine (or cysteine) peptidase inhibitor,
clade A, member 3N
Secreted phosphoprotein 1
T-cell specific GTPase
WW domain containing E3 ubiquitin protein ligase 1*
Genes with up- or down-regulated transcriptions in the liver of Echinococcus multilocularis (E.multilocularis)-infected BALB/c mice are shown in comparison with
noninfected sham-injected control animals (fold increase/decrease).
isotypes with possibly different roles; 3) a parallel course of
cytokines and their related chemokines was highly in favor of their
permanent role to maintain the homing of immune cells at close
proximity of the parasitic vesicles; and 4) at least some of the
components of the immune response were present in the
surrounding liver and were thus involved in a process which was
long considered to be a localized tumor-like event (Figure 6 and
In the present study, we found that IL-12a and TNF-a were
developing in parallel during the different stages of E. multilocularis
infection. After an initial increase, IL-12a and TNF-a expression
decreased dramatically after the 30th day of infection of mice. This
fits well to previous findings, which had indicated a protective role
against E. multilocularis by in vivo treatment with recombinant IL-12
in C57BL/6J mice , while mice KO for TNF-a , as well as
patients with AE treated with a TNF-a inhibitor , had a faster
and more severe course of disease. IL-1b and IL-6 were then
showing up, presumably to sustain the inflammatory response,
with a mirror image of their respective increase all along
infection. The initial peak of IL-6 as early as 2 days post-infection
may be related to the early activation of the acute phase protein
genes in the hepatocytes, disclosed by previous microarray studies
[21,22]. Conversely, the absence of a significant increase of IL-6 at
day 270 probably explains why, despite increased levels of
haptoglobin, a-1 acid glycoprotein, C3 and C4, and ceruloplasmin
in patients with AE, no increase of C-reactive protein (CRP) levels,
typically associated with IL-6 stimulation, is usually observed,
except in cases complicated by bacterial infection. Secretion of the
pro-inflammatory cytokines IL-1b and IL-18 by PBMC of AE
patients had been shown to be reduced in response to E.
multilocularis metacestode vesicles, compared to controls . In
our study in mice, although IL-1b was highly expressed at the
early and middle stage, it subsequently decreased at the late stage
and was not significantly different from control mice at day 180
post-infection and later, a time point which may approximately
represent the disease stage of most patients at diagnosis of AE.
Such selective dynamics of pro-inflammatory cytokine release may
both install and maintain the periparasitic immune infiltrate from
the very early stage of infection on, and also limit its activation and
thus participate in the tolerance process.
In most previous studies, secretion and expression of cytokines,
chemokines, and related factors that govern immune cell-homing
to E. multilocularis infection site were studied in the peripheral blood
of human AE patients , and in lymph node or spleen cells of
experimentally infected mice [13,24,25]; in situ investigations
focussing on the periparasitic infiltrate and the adjacent liver tissue
are virtually lacking. Early expression of IFN-c, as previously
shown in studies on peripheral lymphocytes, was also confirmed in
our longitudinal study of the periparasitic infiltrate; we hypothesize
that it was very likely induced by the early expression of IL-12.
The apparent decrease in IFN-c at day 8 may be due either to a
technical artefact or, more probably, to a temporary inhibition by
IL-4, also markedly expressed at days 2 and 8 p.i.. Sustained
IFNc expression together with the permanent expression of Th1
chemokines, and its negative correlation with TGF-b1 in the
parasitic lesions all along the course of infection, although Th2 and
T-reg cytokines are also permanently expressed, suggests that
IFNc is very important for the persistence of the periparasitic infiltrate
by permanent homing of immune cells and/or inhibition of their
emigration. The decrease of IL-12 after the early stage of disease
could be, at least partly, responsible for the lack of activation of
CD8 T-cell or NK cell cytotoxicity despite the presence of IFN-c
Several concordant observations showed that the PBMCs of AE
patients as well as spleen or lymph node cells of experimentally
infected mice exhibit a markedly and steadily
increasingTh2oriented response characterized by high levels of IL-4, IL-5
andIL10 expression . The results from many studies have clearly
identified IL-4/IL-5/IL-10 as important regulatory cytokines in
parasitic infections, such as infection by Schistosoma mansoni in mice
[28,29] and humans , Schistosoma haematobium , Trichuris
muris , and Trichinella spiralis . In E. granulosus infection,
IL4/IL-5/IL-10 had been found to be predominant in serum
samples of infected individuals ; furthermore, in the peritoneal
cells of experimental mice, i.e. at the site of E. granulosus
establishment, IFN-c was secreted first, at day 3, but as early as
day 5, a Th2-type response, including IL-4 and IL-13 was
stimulated . These results in CE suggest that a Th2-type
response does not impair the establishment of E. granulosus
metacestode, and does not prevent the development of the
pericyst, a characteristic of CE pathology, which, conversely to
AE, limits the progression of the metacestode . In the parasitic
lesions of E. multilocularis-infected mice, we observed a biphasic
curve of IL-4 mRNA expression, with also a very early peak at 28
days. This early peak differed from what is usually reported in E.
multilocularis infection upon investigation of peripheral lymphocytes
stimulated by E. multilocularis antigens . The early local
expression of IL-4 mRNA might be crucial to prime naive
CD4+ T cells into differentiated Th2 type cells , and to prevent
anti-parasite resistance, such as that occurring in most
intermediate hosts, including humans. We hypothesize that early IL-4
mRNA expression is likely induced through the activation of
innate immunity by specific metabolic components of the
metacestode. Such an activation of IL-4 production has actually
been described in vitro under the influence of Echinococcus
components, both from E. multilocularis  and from E. granulosus
. In the present study, we also found a delayed increase of IL-5
and IL-10 in the middle/late stage of E. multilocularis infection.
This delayed increase of IL-5 and IL-10 is matching previous
observations made by others at the late stage of infection, in
human AE [37,38,39] and are in agreement with the data usually
reported from the study of lymphocytes from experimentally
infected mice ; this combined cytokine profile has been
strongly linked to parasite evasion from the host immune response
The discovery of the IL-17 cytokine family has added a new
dimension to the balance of inflammation and tolerance during
parasite infections. The presence of IL-17-secreting CD4+ T
(Th17) lymphocytes correlates with severe hepatic pathology in
murine schistosomiasis . In our study, IL-17, as detected by a
monoclonal antibody directed against the common epitopes of the
protein, was present in cells of the periparasitic infiltrate all along
the course of infection; however, as far as the expression of mRNA
isotypes of the cytokines is concerned, both IL-17A and IL-17F
were increased at the early stage of E. multilocularis infection, and
then decreased at the late stage; they were both positively
correlated with CCL12 and CCL17; however, IL-17A exhibited
a positive correlation with TNF-a, and appeared lower than even
in controls, at the late stage of infection, while IL-17F was also
expressed at low levels, but still higher than controls. This may
indicate that IL-17A was rather protective but quickly inhibited,
while IL-17F was less suppressed with time and may contribute to
both protection and pathogenesis, as reported in human AE
Chemokines are involved in the homing and persistence of
immune cells in inflammatory reactions, especially to infectious
agents [43,44]; they also participate in innate recognition stages of
immunity and may help direct Th1 and Th2 cytokine-producing
cells during the generation of adaptive immunity . There is
also considerable in vitro evidence that cytokines further capitalize
on these molecules by regulating their expression and secretion
and by using them to activate effector cells such as macrophages
and fibroblasts . Conversely, specific suppression of certain
chemokine production and/or function by E. multilocularis
metacestode in AE patients may constitute an additional immune
escape mechanism . We only measured the mRNA expression
of key chemokines, directly related to the main cytokine profiles,
among the multiple components with chemokine activity. But all
Figure 7. Schematic diagram summarizing the pathways of immune response involved in the host-parasite relationship in E.
measured chemokines were significantly expressed at a given stage
of infection. These results confirmed the importance of these
compounds to maintain the granulomatous infiltrate at the
proximity of the metacestode. The courseof Th1-related
chemokines appeared complementary; CXCL 9 was more expressed
when CXCL10 was less expressed, and vice versa, with a mirror
image, as previously described for IL-1 and IL-6. This may
indicate some balance to ensure lymphocyte homing and
persistence in the lesions. Th2-related chemokines were also
permanently expressed: expression of CCL12 and CCL17
followed the course of IL-4, and CCL 8 followed the course of
IL-5. Such changes in chemokine release may prevent pathogenic
inflammation at the late stage. In addition, the microarray
technique revealed a hyper-expression of RANTES (CCL5),
chemotactic for Th1 cells, eosinophils, and basophils . This
finding suggests that this chemokine is also secreted by cells of the
granuloma at the early stage (830 days) when IL-12, IFN-c and
IL-17 secretions are at their maximum. This should consequently
also be explored more in detail in future studies.
The involvement of the adjacent, not directly affected liver
tissue in the immune process of E. multilocularis/host interaction
has received little attention. Recent studies have provided evidence
that the adjacent liver was fully involved in the relationship
between the parasite and its host; these studies have mostly focused
on the proliferation/apoptosis balance  and the involvement of
the TGF-b/Smad system . Our study confirms that other
mediators of the immune reaction and their receptors appear
principally expressed in the liver tissue, thus also in areas not
directly affected by the parasite and the periparasitic granuloma.
In the adjacent periparasitic liver tissue, the expression of the
various cytokines/chemokines was selective: not all cytokines/
chemokines were expressed in the surrounding liver; some seemed
to be specific for the immune cells of the periparasitic infiltrate,
e.g. TNF-a, IL-17F and CCL8, which were not expressed at all in
the liver. The contribution of the surrounding liver tissue,
however, was quite significant for other ones, e.g. IL-12, IFN-c,
IL-4 and IL-17A, at the early stage of infection; CXCL9, IL-4,
IL5, CCL17, at the middle stage; and IL-10 and TGF-b at the late
stage of infection. From our study, which was performed on liver
samples without cell identification, it is difficult to know if such
expression was restricted to cells of the immune response present
in the sinusoids/portal spaces after their homing to the liver, or
was also present in autochthonous liver cells such as Kupffer cells,
stellate cells, or hepatocytes. Precise identification and respective
location will require appropriate studies. Among cytokine
receptors, only those for IL-1 (IL-R1 like), IL-7, IL-13 (IL-13 Ra1), and
IL-17 (IL-17 R) were up-regulated. This indirectly suggests that
the liver was affected by at least one pro-inflammatory cytokine
(IL-1) and one growth factor (IL-7), and by two types of
Thcytokines (Th2 and Th17). However, absence of up-regulation of
IL-6 and TGF-b receptors in hepatic cells is puzzling and has to be
further confirmed using other techniques in the same model.
Materials and Methods
The animal study was performed in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals. The protocol was approved by the Animal
Care and Use Committee and the Ethical Committee of First
Affiliated Hospital of Xinjiang Medical University (20081205-2).
All surgery was performed under sodium pentobarbital anesthesia,
and every effort was made to minimize suffering.
Mice and experimental design
Pathogen-free female BALB/c mice (810-week old) purchased
from the Animal Center of Xinjiang Medical University
(accredited by the ALLLAC) were housed in cages with a 12-h light/dark
cycle and provided with conventional rodent chow and water ad
libitum. All animals received human care in compliance with the
Medical Research Centers guidelines, and animal procedures
were approved by the Animal Care and Use Committee and the
Ethical Committee of First Affiliated Hospital of Xinjiang Medical
University. Echinococcus multilocularis (E. multilocularis) metacestodes
were obtained from intraperitoneal lesions maintained in Meriones
unguiculatus, and 0.1 mL of pooled lesion suspension was injected
into the anterior liver lobe of mice to be experimentally infected.
For each autopsy time-point, eight experimentally infected mice
were used in the E. multilocularis group (n = 8) and compared with
five control mice (n = 5), which received an intra-hepatic injection
of 0.1 mL sterile saline solution into the anterior liver lobe using
the same surgical procedure. Mice were killed at 2 and 8 days p.i.,
and subsequently at 1, 2, 3, 6, 9 and 12 months p.i., respectively.
Tissue sampling of the parasitic lesion and surrounding
granuloma, and of adjacent non-affected (periparasitic)
liver tissue; and histological examination
In E. multilocularis infected mice, liver samples were taken both
from (1) the parasitic lesion (including liver tissue directly adjacent
by 1 mm to the macroscopically visible parasitic lesion,
subsequently designated as parasitic lesion tissue) for qRT-PCR,
histopathology and immunohistochemistry ; and from (2) the
liver tissue relatively close to the lesion (subsequently designated as
periparasitic liver tissue), i.e. starting 2 mm from the
macroscopic changes due to the metacestode/granuloma lesion, thus
avoiding gross contamination of liver tissue by parasitic
E.multilocularis tissue/cells and correspondingly involved infiltrating host
immune cells, for both qRT-PCR and microarray analyses. Tissue
fragments were directly deep-frozen in liquid nitrogen. Control
samples were taken from the same (anterior) liver lobe from
noninfected control mice.
RNA extraction and cDNA synthesis
Lesion and periparasitic liver tissue samples of each mouse
were processed and analyzed separately. Approximately 50 mm3
sized tissue samples from E. multilocularis infected mice or same size
liver tissue samples from control mice were used to extract total
RNA using TRIzol reagent (Invitrogen, Gaithersburg, MD, USA).
The quality of RNA was confirmed by formaldehyde agarose gel
electrophoresis, and the concentration of RNA was determined by
reading the absorbance at 260/280 nm.
cDNA was synthesized from 1 mg of RNA in the presence of
ribonuclease inhibitor (Promega, Shanghai, China), dNTPs,
Oligo(dT) 18 primers, and RevertAidTM M-Mulv reverse
transcriptase in a total of 25 `L reaction mix.
Quantitative real-time RT-PCR
qRT-PCR was run in a thermocycler (iQ5 Bio-Rad, Hercules,
CA, USA) with the SYBR Green PCR premix (Qiagen, Hilden,
Germany) following the manufacturers instructions.
Thermocycling was performed in a final volume of 20 mL containing 2 mL
cDNA and 10 pM of each primer (Table 6). To normalize for
gene expression, mRNA expression of the housekeeping gene
bactin was measured in parallel. For every sample, both the
housekeeping and the target genes were amplified in triplicate
using the following cycle scheme: after initial denaturation of the
samples at 95uC for 1 min, 40 cycles of 95uC for 5 s and 60uC (or
other) for 30 s were performed. Fluorescence was measured in
every cycle, and a melting curve was analyzed after the PCR by
increasing the temperature from 55 to 95uC (0.5uC increments). A
defined single peak was obtained for all amplicons, confirming the
specificity of the amplification.
Microarray data analyses and annotation of gene
RNA extracts from 3 infected and 3 control mice were selected
for array hybridization, corresponding to 30 days, 60 days, 90 days
and 180 days after infection. Total RNA was purified with
NucleospinH RNA Clean-up Kit (Macherey-Nagel, Germany)
and each purified RNA sample isolated from an individual sample
was run on a single microarray. All microarray procedures were
done according to a previously described procedure .
The original microarray data have been uploaded to Gene
Expression Omnibus (GEO) website: http://www.ncbi.nlm.nih.
gov/geo/index.cgi (accession number: GSE24376). All data is
Immunohistochemistry was performed on formalin-fixed,
paraffin-embedded tissue: 4 mm tissue sections were de-paraffinized in
xylene and rehydrated in gradual dilutions of ethanol. Endogenous
peroxidase was blocked with 3% hydrogen peroxide. To increase
staining, sections were pre-treated by microwave heating for
15 min in antigen unmasking solution (pH 6.8, 0.1 M citrate
buffer, Zhongshan Jinqiao Biology Corporation, Beijing). To block
non-specific background, the sections were incubated with
nonimmune goat serum for 30 min. Sections were then incubated
overnight at 4uC with the primary antibody diluted in pH 7.3
phosphate-buffered saline (PBS) (IL-17 1:100 (Santa Cruz
Corporation, CA, USA). After 3 washes in PBS, the sections were
subsequently incubated with horseradish peroxidase conjugated
host-specific secondary antibodies and 3,39-diaminobenzidine was
used as chromogen. Sections were counterstained with
hematoxylin for 5 min, dehydrated, and covered with slips. For all samples,
negative controls consisted of substitution of the isotype-matched
primary antibody with PBS.
Expression of the data and statistical analysis
Immunostaining for IL-17 was semi-quantified by calculating
expression scores that consider both staining intensity and the
percentage of cells stained at a specific range of intensities. A score
of zero indicated the percentage of positive cells ,5%, 1+ = 5
25%, 2+ = 2550%, 3+ = 5075%, 4+.75%. The staining
intensity of each specimen was judged relative to the intensity of a
control slide including an adjacent section stained with an
irrelevant negative control antibody that was matched by species
and isotype to the specimen. Staining of the section labelled with
the negative reagent control was considered as background. A
score of zero indicated no staining relative to background, 1+
= weak staining, 2+ = moderate staining, and 3+ = strong staining.
According to standard pathology practices, staining intensity was
reported at the highest level of intensity observed in all tissue
elements, except the distinctive tissue element for which an
expanded scoring scheme was reported. The expression scores
were calculated by multiplying the percentage of positive cells (0
4) and the staining intensity scores (03). For example: for a
specimen with 30% of positive cells(3+), and a moderate staining
intensity (2+), the expression score was362 = 6. Three
pathologists read the sections and established the scores, and they were
blinded to each others results. Cells with a positive
immunostaining were counted in five random visual fields of 0.95 square mm
each, at initial magnification: 620, for each sample.
All the data were analysed by SPSS 17.0. mRNA expression of
the various cytokines, chemokines, and other components of the
immune response of E. multilocularis infected mice were compared
to the results obtained on the liver samples taken from control
Gene bank accession Primer Sequences
F: 59- TATGGCCCAGACCCTCACA-39
F: 59- GTGTGGAGCAACATGTGGAACTCTA-39
F: 59- GCCAGAGCCACATGCTCCTA-39
mice in the sham-infected liver lobe at the same time point. The
results were presented as means 6 SD. One-way ANOVA and
Students t-test were used to compare the differences between
groups, and Spearmans rho was used to analyse the correlation
coefficients. P,0.05 was considered to indicate statistical
Conceived and designed the experiments: JW RL DAV HW. Performed
the experiments: JW LL GL CZ. Analyzed the data: JW WZ OB BG.
Contributed reagents/materials/analysis tools: LL XL. Wrote the paper:
JW RL DAV BG HW.
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