Acta Tropica 181 (2018) 6–10

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

In vivo treatment with IL-17A attenuates hydatid cyst growth and liver T
fibrogenesis in an experimental model of echinococcosis
⁎
Moussa Labsia, Imene Souflia, Lila Khelifia, Zine-Charaf Amirb, Chafia Touil-Boukoffaa,
a
Laboratory of Cellular and Molecular Biology, Department of Biology, University of Sciences and Technology Houari Boumediene. Algiers-Algeria
b
Department of Anatomy and Pathological Cytology, University Hospital Center Mustapha Pacha, Algiers-Algeria

A R T I C L E I N F O A B S T R A C T

Keywords: We aimed to assess the effect of exogenous Interleukin (IL)-17A in experimental model of echinococcosis. Swiss
Secondary experimental echinococcosis mice were inoculated intra-peritoneally with viable protoscoleces (PSCs). Then, IL-17A was administered at 100,
Interleukin (IL)-17A 125 or 150 pg/mL two weeks after cystic echinococcosis (CE) induction. Cyst development and hepatic damage
Tumor necrosis factor (TNF)-α were macroscopically and histologically analyzed. We observed that in vivo IL-17A treatment at 100, 125, and
iNOS
150 pg/mL, reduced metacestode growth by 72.3%, 93.8%, and 96.9%, respectively. Interestingly an ameli-
Nuclear factor-kβ
oration of liver architecture was noted at 125 pg/mL without toxic effect. In this context, we showed less fibrosis
CD68
Fibrosis reaction and reduced expression of iNOS, TNF-α, NF-κb and CD68 in hepatic parenchyma of treated mice by
125 pg/mL of IL-17A. Collectively, our results indicate an antihydatic effect and immunoprotective properties of
IL-17A and suggest its potential therapeutic value against Echinococcus granulosus infection.

1. Introduction propagating this inflammatory response by releasing proinflammatory

cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6,
Cystic echinococcosis (CE) is a severe chronic helminthic disease and IL-1β, and activating other non-parenchymal liver cells (e.g., en-
that affects both humans and domestic animals (Mandal and dothelial or hepatic stellate cells). Many of these proinflammatory
DebMandal, 2012; Gottstein et al., 2017), and is caused by an infection mediators can activate hepatocystic cell death pathways, and can also
with the larval stage of a parasitic cestode, Echinococcus granulosus. CE activate protective signaling pathways via nuclear factor kappa β (NF-
constitutes a serious public health problem in the Mediterranean basin, κβ). Studies conducted in mice indicate that macrophage activity is
particularly in North Africa (Dakkak, 2010). Moreover, CE is char- largely dependent on the recruitment of monocytes into the liver as
acterized by primary cyst development in the liver and lungs and other precursors of tissue macrophages (Zimmermann et al., 2012).
viscera of the intermediate host. An E. granulosus infection is char- Our current experimental model of secondary echinococcosis is
acterized by the prolonged coexistence of the parasite and host in the based on the development of hydatid cysts in the mouse peritoneal
absence of symptoms, and elicits very little inflammation. However, the cavity following an inoculation with viable protoscoleces (PSCs) (Urrea-
immune response interplay between the parasite and the host is com- Paris et al., 2002; Labsi et al., 2016). During the early infection phase of
plex, encompassing effective parasite-killing immune mechanisms im- experimental echinococcosis (one month), innate immunity may play
plemented by the host. These mechanisms are then modulated by the an important role in controlling parasite growth (Urrea-Paris et al.,
parasite to escape the host immune response (Tamarozzi et al., 2016; 2002; Zhang et al., 2008; Rogan et al., 2015). This early phase is
Gottstein et al., 2017). The variability and severity of the clinical ex- characterized by a T helper (Th)1 dominant immune response (Rogan,
pression of this parasitosis are associated with the duration and in- 1998; Zheng, 2013). The chronic stage of an Echinococcus infection
tensity of infection (Mezioug and Touil-Boukoffa, 2012). occurs once the hydatid cyst is fully formed, which is characterized by a
Echinococcus infection causes an imbalance of the immune response more Th2 and T regulatory-dominant immune response (Touil-Boukoffa
within the hepatic tissue, leading to severe destruction of the archi- et al., 1998; Tamarozzi et al., 2010; Tuxun et al., 2012; Zheng, 2013;
tecture due to intensive inflammatory cell infiltration and the devel- Petrone et al., 2015; Gottstein et al., 2017).
opment of fibrosis (Zhang et al., 2003). Experimental murine models of Our team previously reported elevated levels of Nitric Oxide and
liver injury highlight the involvement of Kupffer cells in initiating and IFN-γ in the serum and PBMC supernatants of patients with E.

⁎
Corresponding author.
E-mail addresses: [email protected] (M. Labsi), imenesoufl[email protected] (I. Soufli), khelifi[email protected] (L. Khelifi), [email protected] (Z.-C. Amir),
touilboukoff[email protected] (C. Touil-Boukoffa).

https://doi.org/10.1016/j.actatropica.2018.01.014
Received 25 June 2017; Received in revised form 18 January 2018; Accepted 23 January 2018
0001-706X/ © 2018 Elsevier B.V. All rights reserved.
M. Labsi et al. Acta Tropica 181 (2018) 6–10

granulosus infections (Touil-Boukoffa et al., 1998; Mezioug and Touil- (Ctrl) received no treatment. The PBS group received an intraperitoneal
Boukoffa, 2012). Moreover, these results are correlated with elevated injection of 500 μL sterile PBS instead of PSCs. The CE and IL-17A/CE
levels of IL-17A and IL-6 (Touil-Boukoffa et al., 1998; Mezioug and groups received an intraperitoneal injection with a suspension of 2000
Touil-Boukoffa, 2012). Thus, it is suggested that together, IL-17A and viable PSCs in 500 μL sterile PBS. The IL-17A(100 pg/mL)/CE group, IL-
IFN-γ exert an immuno-protective effect in hydatidosis and thereby 17A(125 pg/mL)/CE group, and IL-17A(150 pg/mL)/CE group, were
contribute to the host defense mechanism against the parasite. treated with an intravenous administration of a 200 μL IL-17A solution
Based on previous reports (Mezioug and Touil-Boukoffa, 2012; at a concentration of 100, 125, and 150 pg/mL, respectively, beginning
Wang et al., 2017), we aimed to investigate the antihydatic and im- at two weeks post-infection. This study was approved by the National
munoprotective effects of IL-17A on a murine model of echinococcosis. Thematic Agency Research in Health (N∘43-ANDRS-2011).
In this context, we evaluated the effect of IL-17A administration on the All mice were euthanized at three months post-PSC inoculation, at
development of murine echinococcosis and the maintenance of liver which time, the peritoneal cysts were isolated and weighed. Infectivity
function and architecture. Hepatic histological changes and the ex- rate was calculated in percentage by taking the total number of mice
pression of iNOS, NF-κβ, TNF-α, and CD68 were evaluated. infected divided by the total number mice. The larval growth and in-
hibition rate were calculated as described by Ma et al. (2007).
2. Materials and methods
2.3. Histopathological analysis
2.1. Isolation of E. granulosus protoscoleces and initiation of experimental
echinococcosis For the histological examination, small sections of liver (according
to cyst localization) were excised, fixed in 10% buffered formalin,
E. granulosus PSC collection was performed as previously described mounted in paraffin blocks and cut into 2-μm-thick sections. The sec-
(Amri and Touil-Boukoffa, 2015). PSC viability was assessed prior to tions were stained with hematoxylin and eosin (H&E) to assess any
inoculation and determined by 0.1% eosin staining. All samples ex- histological changes and Masson's trichrome staining was used to
hibited viability > 98% at the time of the experiment. Swiss mice were evaluate the level of fibrosis.
inoculated intraperitoneally (i.p.) with 2000 viable PSCs resuspended Histological criteria were based on the degree of architectural tissue
in sterile PBS (Urrea-Paris et al., 2002; Labsi et al., 2016; Khelifi et al., changes and cellular infiltration. Images were captured from each slide
2017). using a digital camera (Casio) attached to a light microscope (Motic)
Two weeks after CE induction, IL-17A (Gibco, life technologies, with 4×, 10×, and 40× objectives. Morphometry was performed
California, USA) was administered to Swiss mice by a single in- using FIJI image processing software following calibration with a
travenous tail injection at doses of 100, 125, and 150 pg/mL. This graduated slide. The extent of tissue fibrosis in the pericystic layer of
choice was established prior to the experiment with a dose-effect study the hepatic hydatid cysts was calculated using area measurements. A
by the administration of IL-17A at 25, 50, 100, 125 and 150 pg/mL to total of 30 photomicrographs were analyzed and quantified for each
infected mice. However, we did not observe any effect on the evolution liver sample. Histopathological diagnoses were performed using a
of echinococcosis, host response, or liver architecture at doses of 25, 50 double blinded fashion.
and 75 pg/mL IL-17A, unlike that exhibited at concentrations of 100,
125 and 150 pg/mL of IL-17A. 2.4. Immunohistochemical procedures

2.2. Experimental design An immunohistochemical study of CD68, NF-κβ, TNF-α, and iNOS
expression was performed on formalin-fixed paraffin-embedded sam-
Female Swiss Albino mice (4–6 weeks old) were purchased from the ples. Indirect immunoperoxidase staining was performed in accordance
Pasteur Institute (Algiers, Algeria). These mice were acclimated for one with the manufacturer’s instructions (DAKO, Denmark A/S). The sec-
week before the start of experiments and maintained under normal tions were incubated with rabbit monoclonal antibodies against mouse
conditions with a 12 h dark/light cycle with ad libitum access to food CD68, NF-κβ/p65 subunit, TNF-α, and iNOS (Santa Cruz
and water. Mice ranging in weight from 21 to 23 g were divided into six Biotechnology, Dallas, TX, USA; diluted 1:100, 1:100, and 1:500, re-
groups (n = 6 mice per group) as shown in Fig. 1. The control group spectively). The detection of primary mouse antibodies was performed

Fig. 1. Experimental design.

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M. Labsi et al. Acta Tropica 181 (2018) 6–10

with EnVision™ FLEX. A chromogen solution of 3,3′-diaminobenzidine CD68, and NF-κβ. An important expression of iNOS, TNF-α, and NF-κβ
(DAB) was added. The sections were counterstained with hematoxylin. was found in the hepatocytes and Kupffer cells from the liver sections of
The slides were covered with Faramount Mounting Medium (S3025, mice in the CE, IL-17A(100 pg/mL)/CE, and IL-17A(150 pg/mL)/CE
DAKO, Denmark A/S) and observed using a standard microscope groups compared to the control group (Table 2). Interestingly, treating
(Motic). Pictures were taken using a digital camera (Casio) at infected mice with 125 pg/mL IL-17A induced a significant decrease in
40 × magnification. the levels of iNOS, TNF-α, and NF-κβ (Table 2).
The percentage of cells positively stained for iNOS, TNF-α, NF-κβ, In addition, an important increase in the number of cells that ex-
and CD68 was scored semi-quantitatively. The quantification of posi- pressed CD68 (Kupffer cells) was clearly observed within the damaged
tive cells was performed using FIJI image processing software following liver tissues of mice from the CE, IL-17A(100 pg/mL)/CE, and IL-
calibration with a graduated slide. The quantification was performed on 17A(150 pg/mL)/CE groups compared to the control and IL-
30 high-power fields in each liver sample by two observers. 17A(125 pg/mL)/CE groups (Table 2).
Moreover, the expression of CD68, iNOS, TNF-α, and NF-κβ was
2.5. Statistical analysis significantly increased in the fibrotic and damaged liver areas of the CE,
IL-17A(100 pg/mL)/CE, and IL-17A(150 pg/mL)/CE groups (Table 2).
A Kruskal–Wallis one-way analysis of variance by a ranks test was
used to compare the treatment effects between all groups. Probability 4. Discussion
values of P ≤ 0.05 were considered to be statistically significant.
The liver is a major site of hydatid disease. Hepatic echinococcosis is
3. Results a life-threatening disease characterized by the establishment of a peri-
parasitic granuloma in the liver and irreversible fibrosis, which results
3.1. The effect of IL-17A treatment on the clinical parameters of in bile duct and vessel obstruction and ultimately liver failure (Beschin
experimental echinococcosis et al., 2013). Currently, the basic approach for the treatment of hydatid
cysts includes surgery and chemotherapy (Brunetti et al., 2010; Nunnari
Three months post-infection, 100% of the animals from the CE et al., 2012). However, operative leakage may lead to the dissemination
group developed cysts in the liver and other intraperitoneal areas. of viable protoscoleces into adjacent tissues and thus, the development
Hydatid cystic liver localization was dominated for all groups. of secondary infection (Moazeni and Nacer, 2010). Therefore, cytokine
Treatment of infected mice with 125 and 150 pg/mL IL-17A sig- use has emerged as alternative therapeutic tool for echinococcosis (Al-
nificantly decreased the infectivity rate (66.67% in mice treated with Qaoud and Abdel-Hafez, 2008; Zhang et al., 2017).
125 and 150 pg/mL IL-17A vs. 100% of mice from the CE group). In In addition to Th1 cytokines (e.g., IL-12 and IFN-γ), Th17 cytokines
contrast, the treatment of mice with 100 pg/mL IL-17A had no effect on (e.g., IL-17A) have been shown to exert an immunoprotective effect in
the infectivity rate. The mean weight of the hydatid cysts was sig- human cystic echinococcosis and could contribute to the host defense
nificantly decreased in both the 125 pg/mL and 150 pg/mL IL-17A mechanism against the parasite (Mezioug and Touil-Boukoffa, 2012).
groups compared with the CE group (*P < .05; Table 1). IL-17A In our study, a murine model of cystic echinococcosis was used to
treatment inhibited cyst growth by 72.3% in mice treated with 100 pg/ examine the effects of IL-17A on hepatic hydatid cyst formation and
mL, by 93.8% in the 125 pg/mL group, and 96.9% in the 150 pg/mL growth with regards to liver histological changes and the local ex-
group (Table 1). pression of iNOS, CD68, TNF-α, and NF-κβ.
The administration of 125 pg/mL IL-17A to infected Swiss mice
3.2. Liver architecture was improved in infected mice treated with 125 pg/ showed a significant inhibitory effect on hydatid cyst development. Our
mL IL-17A data revealed a decrease in hydatid cyst weight in infected mice and
treated with this dose of IL-17A. These observations correlated with a
Histological analysis of the liver isolated from the CE group, IL- reduced fibrotic reaction and an amelioration of the liver architecture.
17A(100 pg/mL)/CE group, and IL-17A(150 pg/mL)/CE group, re- In the same line, it has also been reported that the treatment of a
vealed the proliferation of Kupffer cells, congestion of vein blood, and murine Echinococcus multilocularis infection with exogenous IFN-γ de-
degeneration of hepatocytes with fatty vacuoles (Fig. 2C, D, and F). creases metacestode growth and liver fibrogenesis (Liance et al., 1998).
However, the administration of 125 pg/mL IL-17A to mice with CE Using an experimental model of hepatic Echinococcosis in C57BL/6J
caused an improvement in the histological structure of the liver sections mice, Emery et al. (1998) demonstrated that recombinant IL-12 protects
compared to CE, IL-17A(100 pg/mL)/CE and IL-17A(150 pg/mL)/CE C57BL/6J mice against secondary alveolar echinococcosis via the re-
groups (Fig. 2E). This was accompanied by decreased fibrosis. The ex- duction of the larval growth and the burden of an established infection
tent of fibrosis was quantified, revealing a significant difference be- with Echinococcosis mulilocularis. In this manner, the team of Wang et al.
tween the pericyst of the IL-17A(125 pg/mL)/CE and that of the CE, IL- (2017) reported that the depletion of FoxP3 Tregs may be an option for
17A(100 pg/mL)/CE, and IL-17A(150 pg/mL)/CE groups the development of an immunotherapy against alveolar echinococcosis
(****P < .0001; Fig. 3). Mice from the control and control/PBS groups by activating a Th1/Th17-dominant immune response.
exhibited normal hepatic tissue (Fig. 2A and B). In our model, the concentration of 125 pg/mL of IL-17A appears to
Furthermore, we examined the hepatic expression of iNOS, TNF-α, be an effective dose, with no secondary and no hepatotoxic effects. The

Table 1
Effect of IL17A in an experimental model of echinococcosis.

Experimental group Infectivity Rate (%) Diameter of cysts (mm) Weight of cysts (mg) Number of cysts Mice with hepatic hydatid cysts Inhibition rate (%)

CE group 100 (6/6) 1.9 (13.9) 6.5 (15.2) 10.0 (11.1) 5/6 –
IL-17A (100 pg/mL)/CE group 100 (6/6) 0.7 (9.7) 1.8 (10.4) 9.0 (9.7) 4/6 72.3
IL-17A (125 pg/mL)/CE group 66.7 (4/6) 0.8 (6.0) 0.4 (5.4)* 10.0 (9.0) 4/4 93.8
IL-17A (150 pg/mL)/CE group 66.7 (4/6) 0.8 (6.2) 0.2 (4.6)* 10.0 (9.7) 3/4 96.9

All the cysts observed in all the groups of mice were processed to obtain the parameters reported. Diameter, weight, and number of cysts are presented as median (range). P values from
Kruskal-Wallis test are indicated (*P < .05).
The bold value signifies the effect of IL-17A treatment on the clinical parameters of experimental echinococcosis.

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Fig. 2. Representative photomicrographs (x10) of H&E stained liver tissue.

A: Ctrl group; B: Ctrl/PBS group; C: liver section damaged in CE group; D: liver section heavily damaged in IL-17A(100 pg/mL)/CE group; E: liver section ameliorated in IL-17A(125 pg/
mL)/CE group; F: liver section heavily damaged in IL-17(150 pg/mL)/CE group. (▼): proliferation of kupffer cell; (⇇): congestion of vein blood; (↱): degeneration of hepatocytes with
fatty vacuoles; (v): hepatic vein.

different actions of IL-17A concentrations reported in our study are inflammation, and subsequent liver fibrosis (Tang et al., 2011;
probably related to the complex parasite-host relationship. In our Hammerich et al., 2011).
context, it appears that the concentration of 125 pg/mL is the optimal Interestingly, the treatment of infected mice with 125 pg/mL IL-17A
dose required to be effective in killing the parasitic larvae with pre- induced an important amelioration of the liver architecture, reduced
serving the liver. fibrosis reaction and decreased expression of iNOS, TNF- α, NF-κβ, and
In contrast, the dose of 100 pg/mL would be ineffective against the CD68 in the liver parenchyma.CD68 is a maker of macrophages, a
parasite. In this case, the fibrosis would result from the parasitic action. major source of inducible NO synthase.
In this context, the parasite would not be totally eliminated. It has been shown that macrophages play a key role in the initiation
The dose of 150 pg/mL showed a hepatotoxic effect. The liver fi- and progression of fibrosis (Heymann et al., 2009). Resident macro-
brosis observed, would be related to the excess of IL-17A concentra- phages have shown to play a role in initiating an inflammatory response
tions. In this case, IL-17A would exert a direct inflammatory effect in during tissue injury, while infiltrating monocyte-derived macrophages
host system enhancing injury, especially in hepatic parenchyma. are associated with chronic liver inflammation and fibrogenesis
Accumulating evidence has shown that IL-17A production may favor in (Heymann et al., 2009; Tacke and Zimmermann, 2014). The involve-
various kinds of liver diseases by exacerbation of hepatic steatosis and ment of Kupffer cell-derived cytokine production is underscored by

Fig. 3. Percentage of fibrosis in the pericyst layer of

hepatic hydatid cyst from the CE group, IL-
17A(100 pg/mL)/CE group, IL-17A(125 pg/mL)/CE
group, and IL-17A(150 pg/mL)/CE group. Data are
presented as median with range. p values Kruskal-
Wallis test are indicated (ns: non significatif,
****
P < .0001 vs. pericyst of the CE group).

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M. Labsi et al. Acta Tropica 181 (2018) 6–10

Table 2
Percentages of iNOS, TNF-α, NF-κB, and CD68 expression in hepatic sections of Ctrl group, CE group, IL17A(100 pg/mL)/CE group, IL17A(125 pg/mL)/CE group and IL17A(150 pg/mL)/
CE group.

Antibody expression iNOS in liver sections TNF-α in liver sections NF-κβ in liver sections CD68 in liver sections CD68 in pericyst

Ctrl group 4.6 (38.7) 3.3 (38.6) 3.3 (66.2) 0.2 (20.7) –
CE group 30.0 (135.2)#### 41.9 (128.7)#### 10.3 (123.3)#### 4.8 (113.3)#### 1.9 (91.1)
IL-17A (100 pg/mL)/CE group 14.2 (94.3)** 18.0 (110.2)ns 5.7 (87.8)* 4.3 (116.8)ns 1.1 (64.7)Δ
IL-17A (125 pg/mL)/CE group 4.5 (36.8)**** 3.6 (43.1)**** 1.3 (30.6)**** 0.6 (43.0)**** 0.3 (38.3)‡
IL-17A (150 pg/mL)/CE group 10.9 (72.5)**** 9.2 (56.9)**** 2.6 (69.6)**** 2.0 (83.6)ns 0.8 (47.9)‡

Data are presented as median (range). p values Kruskal-Wallis test are indicated (####p < .0001 vs. Ctrl group, ns not significant vs. liver sections of the CE group, *p < 0.05 vs. liver
sections of the CE group, **p < .005 vs. liver sections of the CE group,****p < .0001 vs. liver sections of the CE group, Δp < .05 vs. pericyst of the CE group, ‡p < .0001 vs. pericyst of
the CE group).

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