Accepted Manuscript

Characterising granuloma regression and liver recovery in a murine model ofschistosomiasis japonica

Candy Chuah, Malcolm K. Jones, Donald P. McManus, Sujeevi K. Nawaratna,Melissa L. Burke, Helen C. Owen, Grant A. Ramm, Geoffrey N. Gobert

PII: S0020-7519(16)00005-9DOI: http://dx.doi.org/10.1016/j.ijpara.2015.12.004Reference: PARA 3831

To appear in: International Journal for Parasitology

Received Date: 26 August 2015Revised Date: 30 November 2015Accepted Date: 7 December 2015

Please cite this article as: Chuah, C., Jones, M.K., McManus, D.P., Nawaratna, S.K., Burke, M.L., Owen, H.C.,Ramm, G.A., Gobert, G.N., Characterising granuloma regression and liver recovery in a murine model ofschistosomiasis japonica, International Journal for Parasitology (2016), doi: http://dx.doi.org/10.1016/j.ijpara.2015.12.004

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting proof before it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Characterising granuloma regression and liver recovery in a murine model of

schistosomiasis japonica

Candy Chuah a,b,c

, Malcolm K. Jones b, Donald P. McManus

a, Sujeevi K. Nawaratna

a, Melissa L.

Burke a,d

, Helen C. Owen b

, Grant A. Ramm a, Geoffrey N. Gobert

a,*

aQIMR Berghofer Medical Research Institute, Brisbane, Qld 4006, Australia

bSchool of Veterinary Sciences, The University of Queensland, Gatton, Qld 4343, Australia

cSchool of Medical Sciences, Universiti Sains Malaysia, 16150, Kelantan, Malaysia

dPresent address: European Molecular Biology Laboratory, European Bioinformatics Institute,

EMBL-EBI, Wellcome Trust Genome Campus, Hinxton CB10 1SD, United Kingdom

* Corresponding author. Tel.: +61 7 3362 0406; fax: +61 7 33620104

E-mail address: Geoffrey.Gobert@qimrberghofer.edu.au

ABSTRACT

For hepatic schistosomiasis the egg-induced granulomatous response and the development

of extensive fibrosis are the main pathologies. We used a Schistosoma japonicum-infected mouse

model to characterise the multi-cellular pathways associated with the recovery from hepatic fibrosis

following clearance of the infection with the anti-schistosomal drug, praziquantel. In the recovering

liver splenomegaly, granuloma density and liver fibrosis were all reduced. Inflammatory cell

infiltration into the liver was evident, and the numbers of neutrophils, eosinophils and macrophages

were significantly decreased. Transcriptomic analysis revealed the up-regulation of fatty acid

metabolism genes and the identification of Peroxisome proliferator activated receptor alpha (PPAR-

α) as the upstream regulator of liver recovery. The aryl hydrocarbon receptor signalling pathway

which regulates xenobiotic metabolism was also differentially up-regulated. These findings provide

a better understanding of the mechanisms associated with the regression of hepatic schistosomiasis.

Keywords: Schistosomiasis; Hepatic fibrosis; Helminth; Transcriptomics; Liver recovery

1. Introduction

Schistosomiasis is a chronic helminth disease of humans caused by digenean trematodes of

the genus Schistosoma. Although this disease has been successfully controlled in many countries, it

is still a major threat to public health as approximately 260 million people in over 78 countries are

infected (World Health Organization, 2015). Chronic infection with the Asiatic Schistosoma

japonicum ranges from mild hypersensitivity reactions to granuloma formation, periportal fibrosis,

portal hypertension, porto-caval shunting, and bleeding from gastrointestinal varices, which may be

fatal (Gryseels et al., 2006; Burke et al., 2009). Pathology is associated with the host CD4+ Th2

response, with IL-4 and IL-13 the dominant cytokines responsible (Wynn et al., 2004). Chemokines

and their receptors have also been shown to play an important role in the development of

schistosome egg-induced granuloma formation (Chuah et al., 2014a). The transcriptional regulation

of hepatic schistosomiasis japonica in mouse models has been investigated during the acute phase

of granuloma formation (Burke et al., 2010a, b; Perry et al., 2011; Chuah et al., 2013); however less

is known regarding the more chronic phases and the events that occur in the liver during recovery

after parasite clearance.

Current efforts to control schistosomiasis rely solely on the drug praziquantel (PZQ) (Xiao

et al., 2009). The pharmacological actions of PZQ on adult schistosomes are considered to be

calcium-dependent (Doenhoff et al., 2008), and the participation of host complement in vivo is also

an important co-factor for parasite elimination (La Flamme et al., 2003). PZQ, in addition to its

anti-helminth effect, is reported to have anti-inflammatory properties (Ribeiro-dos-Santos et al.,

2006). PZQ administration significantly reduces granuloma area, the number of inflammatory cells

within the granulomas (Yang et al., 1984; Huang et al., 2011), and the levels of inflammatory

cytokines in the blood (Silveira-Lemos et al., 2013). PZQ treatment successfully decelerated hepatic

fibrosis in patients suffering from schistosomiasis mansoni (Zwingenberger et al., 1990), and can

also reverse pulmonary hypertension and vascular remodelling in a murine model of Schistosoma

mansoni infection (Crosby et al., 2011). The transcriptional response of the parasite to PZQ has

advanced the understanding of its mode of action (Gobert, 2010). While some insights have been

made in regards to the anti-parasite response of PZQ for both S. japonicum (You et al., 2013) and S.

mansoni (Aragon et al., 2009; Hines-Kay et al., 2012; Kasinathan et al., 2014), less is understood

regarding the impact of the drug on host tissues, particularly in the main organ of pathology, the

liver.

Schistosomiasis is a disease caused predominantly by the host immune response to

schistosome eggs (ova) and the granulomatous reaction they evoke (Pearce and MacDonald, 2002).

Granulomas are formed to destroy eggs and sequester or neutralize otherwise pathogenic egg

antigens but this process also leads to host tissue fibrosis (Wilson et al., 2007). Transforming

growth factor beta (TGF-β) is involved in the process of liver regeneration after partial hepatectomy

(Braun et al., 1988), and it plays a role in hepatocyte proliferation in the regenerating liver

(Thenappan et al., 2010). In schistosomiasis, the regression of hepatic granuloma and the down-

modulation of the Th2 response are thought to be mediated by IL-10 secreting T regulatory cells

(Hesse et al., 2004), and the role of IL-13R�2 in the resolution of fibrosis has been reported

(Chiaramonte et al., 2003). However, the specific mechanisms leading to liver recovery are still not

clear. In this study, we describe the application of a mouse model of S. japonicum treated with PZQ

to allow liver recovery, and the characterisation of these events using histology and by whole

genome microarray analysis. These results provide novel insights on hepatic cellular events during

recovery after hepatic schistosomiasis.

2. Materials and methods

2.1. Ethics statement

All animal studies were conducted with the approval of the Animal Ethics Committee of

QIMR Berghofer Medical Research Institute, Brisbane, Australia.

2.2. Parasites and mouse infections

A total of 42, 4 - 6 week old female C57BL/6 mice (n = 6 per group) were percutaneously

infected with 14 ± 1 S. japonicum cercariae (Chinese mainland, Anhui population). An additional

14 C57BL/6 mice were used as uninfected controls.

2.3. Drug administration

The mice were divided into groups (Fig. 1) designated as: PZQ-treated infected mice (PI; n

= 18, six mice per time point); untreated infected mice (NI; n = 24, six mice per time point); PZQ-

treated uninfected mice (PU; n = 12, four mice per time point); and untreated uninfected mice (NU;

n = 2). A group of six infected mice (from group NI) was euthanised at 6 weeks p.i. before the

commencement of PZQ treatment, to provide baseline data. Mice receiving PZQ treatment (Groups

PI and PU) were orally administered 150, 200, 250, 300 and 350 mg/kg PZQ prepared in 2.5% (v/v)

Cremophor EL (Sigma, USA) for five consecutive days at 7 weeks p.i. Groups NI, PI and PU were

euthanised 3, 6 and 7 weeks post PZQ treatment (10, 13 or 14 weeks post cercarial challenge,

respectively) and the absence of worms was confirmed at sacrifice (see Section 2.4).

2.4. Parasitological, pathological and histological assessment

Adult worms were obtained and counted following perfusion from the intestinal mesenteric

veins, and mouse livers and spleens were collected for assessment of hepatosplenomegaly. The

small lobe of the liver was used for histology (see below), then the remaining tissue was divided for

RNA isolation and egg counts. Hepatic egg burden was calculated as the number of eggs per gram

of liver (EPG) as previously described (Burke et al., 2010a). Faeces were collected 1 week before

PZQ administration to microscopically confirm the presence of parasite eggs and again 1 week after

PZQ treatment to ensure that treatment was effective, as described (You et al., 2012).

The small lobe of liver from each mouse was formalin-fixed 10% (v/v) and paraffin-

embedded, and liver sections were then stained with H&E to assess granuloma density, and

PicroSirius Red for collagen to measure hepatic fibrosis (Perry et al., 2011). Leder and Giemsa

staining measured neutrophil and eosinophil infiltration, while quantitation was carried out by

determining the average number of positive-stained cells over 20 fields at high magnification as

previously described (Burke et al., 2010a). Macrophage recruitment was evaluated by F4/80

immunostaining and quantitated using Aperio’s Spectrum Plus software Positive Pixel Count

Algorithm (Burke et al., 2010b). ���� slides were digitised using the Aperio Slide Scanner (Aperio

Technologies, USA).

2.5. Total RNA isolation

Total RNAs from all liver samples were isolated using Trizol and a RNeasy Mini Kit

(Qiagen, Germany) following the manufacturer’s instructions. Total RNA quantity was measured

using a Nanodrop-1000 spectrophotometer (Nanodrop Technologies, USA) and RNA quality was

assessed using an Agilent Bioanalyzer (Agilent Technologies, USA).

2.6. Microarray analysis

Microarray analyses were performed using Illumina Mouse Ref-8 Version 2 whole genome

expression arrays as previously reported (Chuah et al., 2013). Details of cRNA synthesis and Whole

Genome Microarray Hybridisation, Feature Extraction and data analysis, and Ingenuity Pathway

Analysis (IPA), are outlined in Supplementary Data S1. All gene expression data have been

submitted to Gene Expression Omnibus (National Center for Biotechnology Information (NCBI),

USA) and are publicly available (Series Accession Number, GSE59276).

2.7. Quantitative real-time PCR (qRT-PCR)

cDNA was synthesized using a Quantitect Reverse Transcription kit (Qiagen) and cDNA

concentration quantified using a Nanodrop-1000 spectrophotometer (Nanodrop Technologies).

qRT-PCR was performed using SYBR Green master mix (Applied Biosystems, USA) on a Corbett

Rotor Gene 6000 (Corbett Life Sciences, Australia). Hypoxanthine phosphoribosyltranferase

(HPRT) was used as a housekeeping gene as described (Dheda et al., 2004). Quantitative real-time

PCR (qRT-PCR) was used to validate a subset of microarray data representing those transcripts that

were significantly expressed (up- or down-regulated) by microarray analysis. Primers for qRT-PCR

were designed using Primer 3 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi)

and are listed in Supplementary Table S1.

2.8. Immunohistochemistry

The Ki67 (a marker for cellular proliferation) and tissue inhibitor of metalloproteinase-1

(TIMP1) proteins were immunolocalised using paraffin-embedded section described in Section 2.4.

Full details of the processing procedures are presented in Supplementary Data S1. Two analysis

strategies were used for quantitation of staining as described below.

2.8.1. Semi-quantitative analysis

Hepatocytes and biliary epithelium over five randomly chosen high power fields in each

section were assessed for Ki67 staining. The entire section was also screened for TIMP1 staining

and areas containing positively staining cells were characterised. This semi-quantitative survey was

performed by a veterinary pathologist (co-author Helen C. Owen), who was blinded to the

experimental groupings.

2.8.2. Quantitative analysis

Aperio images were analysed using the Aperio Imagescope v11.1.2.760 software. Positive

Pixel Count V9 algorithm was optimised to count TIMP1 stained cells. Nuclear algorithm V9 was

used to quantitate cells stained with Ki67. The percentage of positive cells was calculated by

comparing the strong positive cells to the total number of positive and negative cells. Data were

analysed using GraphPad Prism Version 6.0 software, with significance between treatment groups

and weeks determined using One-way ANOVA (P ≤0.05).

2.9. Statistical analyses

Statistical analyses of histological and pathological data were performed using GraphPad

Prism Version 6.0 (GraphPad Software, USA). Changes in parasitological and histological data of

the untreated group were assessed by one-way ANOVA, whereas two-way ANOVA (P ≤0.05) was

used to examine changes in the PZQ-treated infected group (Group PI) compared with the untreated

infected group (Group NI) and the PZQ-treated uninfected group (Group PU). The correlation

between microarray data and qRT-PCR was measured using Spearman’s correlation in GraphPad

Prism Version 6.0 as previously reported (Morey et al., 2006).

3. Results

3.1. General histopathological features

Uninfected mice (Groups PU and NU) showed normal hepatic lobular architecture (Fig. 2A).

Schistosome eggs observed in the liver at 6 weeks p.i. were surrounded by a dense population of

neutrophils and infiltrative macrophages within a band of proliferating fibrovascular tissue (Fig.

2B). At 10 weeks p.i., eggs were surrounded by a dense population of macrophages interspersed

with a few aggregates of neutrophils with a further ring of lymphocytes at the periphery (Fig. 2C).

At 13 and 14 weeks p.i., most eggs were calcified and surrounded by varying proportions of

lymphocytes, fibrosis in a collagenous stroma and macrophages including multinucleated giant cells

(Fig. 2D - E). The granulomas of PZQ-treated, infected mice (Group PI) predominantly contained

remnants of egg shells, presented less cellular infiltrates and were surrounded by concentric bands

of fibrous tissue (Fig. 2F – H).

3.2. Parasitological and histological analyses

Schistosoma japonicum adult worm pairs were present in each infected mouse (Fig. 3A).

Positive infections were confirmed prior to PZQ treatment by copro-microscopic examination. PZQ

administration successfully eliminated all adult parasites (P ≤0.001), as determined by faecal egg

examination and eventual worm counts at the time of host sacrifice and perfusion. Livers of

untreated, infected mice (Group NI) attained the highest weight at 10 week p.i., and decreased

significantly at week 14 p.i. (P ≤0.05) (Fig. 3B). There were no significant liver weight differences

in untreated, infected mice (Group NI) compared with PZQ-treated, infected mice (Group PI) at any

time points. Spleen weights of untreated, infected mice (Group NI) increased significantly from 6

weeks p.i. onwards (P ≤0.001). There was a significant reduction in the spleen weight of PZQ-

treated, infected mice (Group PI) 3, 6, and 7 weeks after PZQ treatment compared with untreated,

infected mice (Group NI) (P ≤0.0001). The spleen weight of PZQ-treated, infected mice (Group PI)

and PZQ-treated, uninfected controls (Group PU) did not differ significantly after 6 and 7 weeks

post PZQ treatment. The data of both liver and spleen weights of untreated, uninfected controls

(Group NU) at 6 weeks are also included (Fig. 3). Both the liver and spleen weights of untreated,

infected mice (Group NI) at 6 weeks p.i. increased significantly compared with the untreated,

uninfected controls (Group NU) at same time point (P ≤0.05).

Hepatic egg burden increased in untreated, infected mice (Group NI) from 6 weeks p.i.

onwards (Fig. 3D) (P ≤0.01), and increased significantly compared with the untreated, uninfected

controls (Group NU) (P ≤0.01). There was no significant difference in the number of eggs in the

liver of PZQ-treated, infected (Group PI) or untreated, infected mice (Group NI) at any time point.

Higher numbers of egg clusters were present in the infected liver of untreated mice (Group NI) at

10 weeks p.i. compared with 6 week p.i. (P ≤0.001), and these decreased significantly thereafter at

13 weeks p.i. (P ≤0.05) (Fig. 3C). Egg clustering also increased significantly in the untreated,

infected mice (Group NI) at 6 weeks p.i. compared with the untreated, uninfected controls (Group

NU) (P ≤0.01). The distribution of egg clusters in PZQ-treated, infected mice (Group PI) was

reduced after 7 weeks of PZQ treatment compared with the untreated, infected group (Group NI) (P

≤0.01), and no significant difference was observed in egg clustering of PZQ-treated, infected

(Group PI) or PZQ-treated, uninfected mice (Group PU) after 6 and 7 weeks of PZQ treatment.

Granuloma density (Fig. 4A – C), the degree of fibrosis (Fig. 4D – F), macrophage (Fig. 4G

– I), neutrophil (Fig. 4J – L) and eosinophil infiltration (Fig. 4M – O) in untreated, infected mice

(Group NI), PZQ-treated, infected mice (Group PI) and PZQ-treated, uninfected mice (Group PU)

were assessed and presented. Granuloma density of untreated, infected mice (Group NI) increased

significantly at 6 weeks p.i. compared with the untreated, uninfected mice (Group NU) (P ≤0.001)

(Fig. 5A). Granuloma density was significantly greater in untreated, infected mice (Group NI)

compared with PZQ-treated, infected mice (Group PI) (P ≤0.05). The area of granulomas in the

PZQ-treated, infected mice (Group PI) did not differ significantly from the PZQ-treated, uninfected

mice (Group PU). Fibrosis, as indicated by collagen staining, was first observed in mature

granulomas at 6 weeks p.i. and was also prominent around the portal vein of the liver. The extent of

fibrosis in the infected livers of untreated mice (Group NI) was higher at 10 - 14 weeks p.i.

compared with 6 weeks p.i. (P ≤0.001) (Fig. 5B). Fibrosis of this group was also significantly

higher at 6 weeks p.i. compared with the untreated, uninfected mice at a similar time-point (Group

NU) (P ≤0.01). Fibrosis decreased from 10 – 14 weeks p.i. but this change was not significant. The

fibrotic response in PZQ-treated, infected mice (Group PI) was reduced significantly compared with

that of untreated, infected mice (Group NI) after 3 weeks of PZQ treatment (P ≤0.01). The degree

of fibrosis between PZQ-treated, infected (Group PI) and PZQ-treated, uninfected mice (Group PU)

did not differ significantly after 6 and 7 weeks of PZQ treatment.

F4/80 staining for macrophages indicated infiltration in the infected livers of untreated mice

(Group NI) peaked at week 10 p.i. (Fig. 5C). Macrophage infiltration into untreated, infected livers

(Group NI) 6 weeks p.i. was significantly greater compared with untreated, uninfected mice (Group

NU) (P ≤0.05) at the exact time point. The infiltration of macrophages to the PZQ-treated, infected

livers (Group PI) decreased significantly compared to the untreated, uninfected livers (Group NU)

(P ≤0.05). There was no significant difference in the numbers of macrophages in the PZQ-treated,

infected (Group PI) or PZQ-treated, uninfected livers (Group PU) at any time point.

The extent of neutrophil infiltration in the infected liver of untreated mice (Group NI)

peaked at 10 weeks p.i. and decreased thereafter, (P ≤0.01) (Fig. 5D). Neutrophil infiltration of the

untreated, infected mice (Group NI) at 6 weeks p.i. was significantly higher compared with the

untreated, uninfected mice (Group NU) (P ≤0.001). The recruitment of neutrophils to the PZQ-

treated, infected liver (Group PI) decreased significantly after PZQ treatment,compared with the

means of untreated, infected livers (Group NI) (P ≤0.0001). There was no significant difference in

the numbers of neutrophils in the PZQ-treated, infected (Group PI) or PZQ-treated, uninfected

livers (Group PU) after 6 and 7 weeks of PZQ treatment.

Eosinophil infiltration peaked at 10 weeks p.i. and did not change in numbers thereafter (P

≤0.05) (Fig. 5E). The infiltration of eosinophils to the infected livers of untreated mice (Group NI)

after 6 weeks p.i. was significantly higher than the untreated, uninfected mice (Group NU) (P

≤0.001). Eosinophil numbers in the PZQ-treated, infected liver (Group PI) were significantly lower

than in untreated, infected livers (Group NI) after 7 weeks of PZQ treatment (P ≤0.05). There was

no difference in eosinophil numbers between PZQ-treated, infected (Group PI) and PZQ-treated,

uninfected mice (Group PU) after 6 and 7 weeks of PZQ treatment.

3.3. Microarray analysis: data normalisation and filtration

During the S. japonicum infection (6 to 14 weeks p.i.) normalised gene expression data for

each of the 25,697 genes on the Illumina microarray were filtered for significant signal and

normalised to untreated, uninfected controls (Group NU) reducing the data set to 11,872 genes

(Supplementary Table S2). Of these, 1,830 genes (Supplementary Table S3) were shown to be

differentially expressed with a ± two-fold change in at least one individual sample at each time

point (one - way ANOVA, P ≤0.05). Transcriptional changes in the PZQ-treated, infected liver

(Group PI) after 3, 6, and 7 weeks of PZQ treatment were examined by normalising the 25,697

genes on the array platform to each of the untreated, infected samples (Group NI) at the same time-

points. This reduced the data set to 516, 402 and 1,099 differentially expressed genes

(Supplementary Table S4) with a ± two-fold change after 3, 6 and 7 weeks of PZQ treatment,

respectively. For the assessment of gene changes due to PZQ alone, data for PZQ-treated,

uninfected mice (Group PU) were filtered and normalised to the uninfected controls without PZQ

treatment (Group NU). This reduced the data list from 25,697 to 10,758 genes, of which seven

genes were shown to be significantly up-regulated with a ± 1.5-fold change (Supplementary Table

S5).

3.4. Genes differentially up-regulated during progression of S. japonicum infection (Group NI)

IPA revealed that genes significantly up-regulated more than two-fold (P ≤0.05) during the

chronic progression of S. japonicum infection compared with untreated, uninfected livers (Group

NU), were primarily related to IL-8 signaling (P value 4.79E-07, see Table 1 and Supplementary

Data S2) and the dendritic cell (DCs) maturation pathway (P value 1.91E-07, see Table 2 and

Supplementary Data S2). Expression of IL-8 associated genes included Myeloperoxidase (MPO),

Matrix Metalloproteinase 9 (MMP9), Myosin Light Chain Regulatory B (MYLC2B), and Arrestin

Beta 2 (ARRB2) with higher expression at 10 or 14 weeks p.i., paralleling increased granuloma

density and collagen deposition at these time points.

Genes associated with DC maturation pathways (Table 2) showed peaked expression at 6

weeks p.i. and included genes such as Fc receptors (FCGR3A, FCER1G, FCGR2A, FCER1G), IL 1

Beta (IL-1β), TYRO Protein Tyrosine Kinase Binding Protein (TYROBP), Phosphoinositide-3-

Kinase, Regulatory Subunit 1 (PIK3R1), Toll-like Receptor 2 (TLR2), Intercellular Adhesion

Molecule 1 (ICAM1) and IL 1 Receptor Antagonist (IL1RN).

Other canonical pathways indentified by IPA in the liver of acute to chronic mice (Group

NI, from 6 to 14 weeks p.i.) included LPS/IL-1 Mediated Inhibition of RXR Function (P value

3.16E-15), Acute Phase Response Signaling (P value 5.01E-14), Xenobiotic Metabolism Signaling

(P value 2.00E-12), Aryl Hydrocarbon Receptor Signaling (P value 2.00E-11), FXR/RXR

Activation (P value 2.51E-11), Fatty Acid oxidation I (P value 7.76E-10), Oxidative

Phosphorylation (P value 1.07E-09), Serotonin Degradation (P value 2.63E-09) and Leukocyte

Extravasation Signaling (P value 3.02E-09) (see Supplementary Data S2).

Gene expression related to hepatocyte growth was down-regulated during acute and chronic

disease (Group NI, from 6 to 14 weeks p.i.) relative to untreated, uninfected controls (Group NU)

included HGFAC: hepatocyte growth factor activator (-2.66, -2.6, -1.79, -3.24 fold; weeks 6, 10, 13,

14) and PCBD2: dimerization cofactor of hepatocyte nuclear factor 1 alpha 2 (-1.79, -1.88, -1.66, -

2.09 fold; weeks 6, 10, 13, 14) (see entary Table S3). TIMP1 (17.48, 10.89, 6.41, 10.24 fold; weeks

6, 10, 13, 14) a gene associate with tissue remodelling was elevated during the acute and chronic

disease (Group NI, from 6 to 14 weeks p.i.) relative to untreated, uninfected controls (Group NU)

peaking at 17.5 fold at 6 weeks p.i. (see Supplementary Table S3).

3.5. Genes differentially up-regulated in PZQ-treated, S. japonicum-infected mice (Group PI)

compared with untreated, S. japonicum-infected mice model (Group NI)

Peroxisome proliferator activated receptor alpha (PPAR-α) was identified by IPA (P value

3.66E-63, see Supplementary Data S3) as an “activated” upstream regulator of the recovery phase

of the liver after 3, 6 and 7 weeks of PZQ treatment. PPAR-α is a ligand-activated transcription

factor that plays critical role in the regulation of hepatic fatty acid metabolism (Crabb et al., 2004).

The identification of this upstream regulator enables the understanding of the series of

transcriptional cascades and biological activities occurring in the recovering liver treated with PZQ.

Transcriptomic analyses showed that the top highly up-regulated genes 3, 6 and 7 weeks

post PZQ treatment were mainly associated with fatty acid and xenobiotic metabolism with peak

expression at 7 weeks post-treatment (Table 3), although higher gene expression past this time point

cannot be discounted. Fatty acid-related genes (Canonical pathway Fatty Acid -oxidation I P value

7.59E-10, see Supplementary Data S3) included Acetyl-Coenzyme A Acyltransferase 1B

(ACAA1B), Acyl-CoA Synthetase Long-chain Family Member 1 (ACSL1), Aldehyde

Dehydrogenase Family 3, Subfamily A2 (ALDH3A2), 3-Hydroxyacyl Coenzyme A Dehydrogenase

(EHHADH), Solute Carrier Family 27, Member 2 (SLC27A2), and Cytochrome P450 family

(CYP2B9, CYP2A12, CYP2D26).

Genes associated with xenobiotic metabolism (canonical pathway P value 2.00E-12, see

Supplementary Data S3) including Cytochrome P450 family (CYP2B9, CYP2A12, CYP2C29,

CYP2C37, CYP2C50, CYP2D26, CYP3A25, CYP2F2), Glutathione S-Transferase (GSTT1, GSTT2,

GSTT3), ALDH3A2, Dihydrodiol Dehydrogenase (DHDH), Flavin containing Monooxygenase 1

(FMO1), Monoamine Oxidase B (MAOB), Sulfotransferase Family 1D, Member 1 (SULT1D1), and

UDP Glucuronosyltransferase family (UGT2A3, UGT2B1, UGT1A10), showed peak expression

(within the time frame examined) at 7 weeks post treatment, indicating that the PZQ-treated liver

slowly attained normal hepatic function. The metabolic activity of PZQ-treated liver was suggested

to have returned to normal level (basal uninfected controls) as when the data of PZQ-treated,

infected liver (Group PI) was normalised against PZQ-treated, uninfected liver (Group PU), these

metabolic genes did not differ significantly.

Expression of most genes peaked (within the time frame examined) 7 weeks post treatment,

indicating that this time point is critical in the recovery process. IPA identified enrichment of aryl

hydrocarbon receptor (AhR) signaling (P value 3.3E-10, see Supplementary Data S3) after 7 weeks

of PZQ treatment compared with the untreated, infected group at the same time point (Table 4).

This receptor is involved in the regulation of enzymes responsible for xenobiotic metabolism

(Ramadoss et al., 2005).

Other canonical pathways identified by IPA (see Supplementary Data S3) included LPS/IL-

1 Mediated Inhibition of RXR Function (P value 1.00E-18), Xenobiotic Metabolism Signaling

(P value 2.00E-12), Nicotine Degradation II (P value 3.98E-12), FXR/RXR Activation (P value

5.01E-12), LXR/RXR Activation (P value 1.00E-11), Serotonin Degradation (P value 1.58E-11),

Superpathway of Melatonin Degradation (P value 3.16E-11), Glutathione-mediated Detoxification

(P value 1.70E-10), Nicotine Degradation III (P value2.09E-10) and Acute Phase Response

Signaling (P value 2.95E-10).

In contrast to acute/chronic schistosomiasis (Group NI, 6 to 14 weeks p.i.), during disease

recovery genes related to hepatocyte growth were up-regulated included HGFAC (2.28 fold; 7

weeks post PZQ treatment) and PCBD2 (2.18 fold; 7 weeks post PZQ treatment) (see

Supplementary Table S4). TIMP1 (-4.07, -4.25, -7.63 fold; 3, 6, 7 weeks post PZQ treatment), a

gene associated with tissue remodelling was reduced during tissue recovery (relative to infected

time course matches) with -7.7 fold evident at 7 weeks post PZQ administration (see Supplementary

Table S4).

3.6. Genes differentially up-regulated in PZQ-treated, uninfected mice (Group PU) compared with

untreated, uninfected mice (Group NU)

No genes were differentially up-regulated by more than two-fold in the PZQ-treated,

uninfected mice (Group PU) compared with the untreated, uninfected mice (Group NU). The

following genes were down-regulated greater than two-fold at 3 weeks post-treatment but all

returned above that cut off 6 and 7 weeks post treatment: Bach1 (BTB and CNC homology 1: -2.02,

1.07, 1.07 fold; 3, 6, 7 weeks post PZQ treatment), Ddx6 (DEAD box polypeptide 6: -2.47, 1.01, -

1.02 fold; 3, 6, 7 weeks post PZQ treatment) and Hpn (hepsin : -2.95, -1.87, -1.82 fold; 3, 6, 7

weeks post PZQ treatment). This finding indicated that PZQ did not exert any significant

transcriptional effects on healthy controls beyond 3 weeks post-drug administration.

3.7. qRT-PCR validation

The expression patterns of a subset of genes (S100A9, MPO, CYP2B9, CYP2A5, CYP8B1,

CYP4A14, FMO3 and INMT) selected from the microarray analysis was validated by qRT-PCR

(Supplementary Fig. S1). There was a significant correlation between the expression levels obtained

by qRT-PCR and microarray data (Pearson’s correlation, r = 0.77; P <0.0001).

3.8. Immunohistochemistry

Wound healing and regeneration of the liver in response to PZQ treatment was investigated

by assessing the expression of TIMP-1 (matrix remodelling) and Ki67 (liver cell proliferation) by

immunohistochemistry, using computer analysis quantitation and a semi-quantitative and qualitative

survey by a blinded veterinary pathologist.

3.8.1. Semi-quantitative survey

Ki67 was predominantly expressed by hepatocytes (Fig. 6A - D). In the PZQ-treated,

infected livers (Group PI), small numbers of lymphocytes in the granulomas and less commonly

lymphocytes in the remaining parenchyma were also positive. Cells demonstrated mild to moderate

staining intensity. The average numbers of cells staining per high power field were elevated in the

untreated, infected samples at 6 weeks p.i. (Fig. 6B) and were also high in the untreated, infected

livers at 10 to 14 weeks p.i. (Fig. 6C). Livers from PZQ-treated, infected animals had lower

numbers of cells staining per high power field than the PZQ-treated, uninfected controls (Fig. 6D).

TIMP1 was predominantly expressed by mononuclear cells (lymphocytes, macrophages and plasma

cells) at the periphery of the granulomas. Bile ductule epithelial cells also stained positively with

TIMP 1 in untreated, infected livers at 10 to 14 weeks p.i. (Fig. 6G). In the untreated, infected livers

of 6 weeks p.i. (Fig. 6F), the granulomas were sometimes intermittently rimmed by a band of

mononuclear cells several cells thick. Up to 50% of these cells were positive. The highest number

of positively staining cells was noted in the untreated, infected livers of 10 to 14 weeks p.i. (Fig.

6G). Granulomas were often intermittently to completely rimmed by a band of mononuclear cells

15-30 cells thick, with up to 70% of these cells being positive. Distinct cellular staining was also

noted in elongated myofibroblast cells located predominantly along the inner region of the TIMP1

positive band. In the PZQ-treated, infected livers (Fig. 6H), granulomas were sometimes

intermittently rimmed by a thin band of mononuclear cells several cells thick, with positively

staining cells scattered throughout this area.

3.8.2. Quantitative analysis

As shown in Fig. 7 the immunohistochemical expression of Ki67 and TIMP1 was

quantitated in untreated, infected livers (Group NI), PZQ-treated, infected livers (Group PI) and

PZQ-treated, uninfected livers (Group PU). Ki67 a marker for cellular proliferation was

undetectable (<0.5%) in PZQ-treated, uninfected liver sections, but was elevated to 11.1% positive

cells in the untreated, infected livers of 6 weeks p.i., and continued to rise during 13 and 14 weeks

p.i. reaching 34.5%. After elimination of the infection the levels of Ki67 dropped to low but still

detectible levels (1.8 - 3.5%). The level of TIMP1 labelling was low in PZQ-treated, uninfected

tissues up to 0.5%, rising to 1.2% in the untreated, infected livers of 6 weeks p.i., but increasing

substantially to 3.3% during 13 and 14 weeks p.i. In the PZQ-treated, infected livers, TIMP1 was

more prominent early at week 10 at 2% but dropped to 0.6 - 0.9% at weeks 13 and 14, respectively.

4. Discussion

While previous investigations have focused mainly on the parasitological and

histopathological changes of the liver after the elimination of a schistosome infection (el-Badrawy

et al., 1988; Huang et al., 2011), the differential gene expression and pathways involved in the

hepatic recovery process have not been reported. In this paper, we employed whole genome

transcriptomic analyses to present a comprehensive overview of the hepatic gene signalling

pathways and the associated mechanisms occurring during the progression of a murine S. japonicum

infection, and the subsequent recovery events in the liver following successful PZQ treatment. The

gene expression changes that we present represent two potential changes in the cellular and

transcriptional profile of the whole organ. While both the transcriptional activity of individual cell

types may be altered, also the relative abundance of cellular populations may be altered as

demonstrated by the quantitative immunohistochemistry performed.

Progression of a chronic S. japonicum infection was significantly associated with the up-

regulation of genes related to IL-8 signalling. IL-8, also known as CXCL8, is a potent neutrophil

chemoattractant (de Oliveira et al., 2013). It has been reported previously that IL-8 responses are

induced in Schistosoma haematobium-infected children (van der Kleij et al., 2004), and the level of

IL-8 was shown to be significantly higher in S. mansoni-infected individuals compared with those

receiving PZQ treatment (Erikstrup et al., 2008). Similarly, infections with S. japonicum elicited an

increase in IL-8 levels in human skin (He et al., 2002). In addition, the transcriptional level of IL-8

has been reported as being up-regulated in human neutrophils stimulated by S. japonicum eggs

(Chuah et al., 2014b). Although the gene encoding IL-8 is not present in the mouse genome,

possibly due to an evolutionary duplication event (Modi and Yoshimura, 1999), it was suggested

that the murine chemokines CXCL1, CXCL2 and CXCL5 may act as the functional homologues of

IL-8 (Rovai et al., 1998; Singer and Sansonetti, 2004; Hol et al., 2010). The up-regulation of

CXCL1 during the chronic progression of S. japonicum infection reported here appears to play a

similar role, and may reflect the involvement of the IL-8 signalling pathway in the pathogenesis of

S. japonicum infection. A recent ex vivo study of hepatic schistosomiasis, using synchronised S.

japonicum soluble egg antigen exposure over 48 h, reported the transcriptional and protein up-

regulation of CXCL1, CXCL2 and IRAK3, supporting the importance of IL-8 related responses to S.

japonicum egg antigen (Gobert et al., 2015). The strong neutrophilic response seen in the livers of

infected hosts is also reflected in the lungs of animal models of migrating schistosomula both from

synchronised intravenous injections (von Lichtenberg et al., 1977), and nature infections (Burke et

al., 2011), indicating that proteins released by other stages have a distinct immunological response

to the host.

Genes related to the DC maturation pathway were significantly up-regulated at 6 weeks p.i.,

and progressively down-regulated after this time-point. DCs are important messengers between the

innate and adaptive immune system, playing a critical role in terms of antigen presentation, and are

known to be involved in directing the polarization of the Th cellular response in schistosomiasis.

The soluble egg antigens of S. mansoni have been shown to direct DCs to promote the

differentiation of naïve Th cell towards a Th2 phenotype (van Liempt et al., 2007; Everts et al.,

2009; Steinfelder et al., 2009). Lacto-N-fucopentose III (LNFPIII), a molecule present on

schistosome egg glycoproteins, was also reported to drive DC maturation via a Toll-like receptor 4

(TLR4)-dependent pathway (Thomas et al., 2003). Here, we suggest that DC maturation is part of

the process occurring during the early stage of schistosomiasis japonica. Again in the ex vivo model

of hepatic schistosomiasis japonica mentioned earlier, IL-1β and TLR-2 up-regulation was a feature

of the first 48 h after exposure to egg antigen (Gobert et al., 2015), which supports the findings in

the current study .

Analysis of the recovering liver revealed PPAR-α as the upstream regulator of observed

transcriptional changes. PPAR-α, the first identified PPAR receptor (Issemann and Green, 1990),

has an elevated transcriptional expression in tissues with high metabolic rates (Burri et al., 2010).

The receptor PPAR-α is a ligand-activated transcription factor crucial for regulation of hepatic fatty

acid metabolism and homeostasis (Desvergne et al., 2004); and in rodents PPAR-α gene expression

is highest in the liver, followed by heart and kidney (Braissant et al., 1996). PPAR-α-null mice

suffering from a severe impairment in hepatic fatty acid oxidation, resulted in elevated levels of

fatty acids and a fatty liver phenotype (Kersten et al., 1999). Activation of PPAR-α also leads to the

up-regulation of genes involved in fatty acid transport, binding and activation (Rakhshandehroo et

al., 2010). PPAR-α activation and the increased transcription of genes related to fatty acid

metabolism observed in our study may thus reflect the downstream mechanism and pathway

occurring in the liver undergoing recovery after schistosomiasis. Furthermore, PPAR-α is also

involved in wound healing and repair, with liver regeneration in PPAR-α-null mice found to be

delayed and impaired following partial hepatectomy (Anderson et al., 2002). Using murine models

PPAR-α was shown to regulate hepatic neutrophil infiltration in ischemic liver injury (Okaya and

Lentsch, 2004), while PPAR-α activation also reduced portal hypertension and liver fibrosis in

carbon tetrachloride (CCl4)-induced cirrhotic models (Rodriguez-Vilarrupla et al., 2012). No role

for PPAR-α has been reported for hepatic schistosomiasis, and the observations we make in

reference to treated and untreated infected mice are at this stage unclear but worthy of future

investigations. While it is known that PPAR-α plays a critical role in regulation of hepatic fatty acid

metabolism, it is unclear how this is related to Schistosoma infection or the effects of PZQ. It is

likely that some other, as yet unknown, aspect of PPAR-α function may be key.

While decreased proliferation of hepatocytes was indicated by reduced gene expression of

PCBD2 and HGFAC during disease progression, and the classical regulator hepatic nuclear factor

4, alpha (Hnf4a) (Walesky et al., 2013) was unchanged (<2 fold) during disease recovery, the

opposite pattern was noted for PCBD2 and HGFAC. This was investigated further by the use of

Ki67 staining, a classic nuclear marker for cellular proliferation (Bonzo et al., 2012), where

elevated proliferation was noted during acute and chronic disease, and reduced but still higher than

uninfected controls, following PZQ treatment and subsequent tissue remodelling. These data show

that the proliferation of liver cells is heightened during hepatic schistosomiasis and granuloma

formation. Furthermore, while reduced during the liver recovery phase, even 3 weeks after recovery

proliferation was still present above basal levels. The examination of earlier time points after

parasite clearance may demonstrate a greater role for cellular proliferation in the recovering liver.

The role of TIMP1 was explored both from analysis of transcriptional data and also

immunohistochemistry. TIMP1 has a clear role in inhibiting various metalloproteinases (MMPs)

(Giannandrea and Parks, 2014) that normally act to degrade extracellular matrix. TIMP-1 protein

expression was low in normal liver but during the acute and chronic phases of hepatic

schistosomiasis it was dramatically increased before being reduced in the recovering liver following

PZQ treatment. These results suggest that with TIMP-1 protein expression significantly increased at

the periphery of the granuloma, matrix resorption would be inhibited by TIMP-1 binding to MMPs

and suppressing their activity that would normally degrade fibrillar collagens, thus facilitating

collagen deposition. One possible mechanism associated with PZQ treatment follows, the level of

TIMP-1 is reduced both through down regulation of gene expression and reduction of the cellular

population, resulting in increased MMP activity, thus degrading the fibrosis surrounding the

granuloma, permitting wound healing and liver mass restitution. Past studies have underplayed the

role of TIMP1 and MMPs in hepatic granuloma formation using S. mansoni as a model (Vaillant et

al., 2001). In previous studies, while increases in TIMP1, TIMP2 and MMP9 gene expression were

evident during disease progression, the use of TIMP1 and TIMP2 knockout mice failed to

demonstrate a functional correlation to disease severity in schistosomiasis mansoni (Vaillant et al.,

2001).

As the main organ involved in drug and toxin removal, the liver plays an important role in

controlling the accumulation of a wide range of toxic substances by converting them into inactive

metabolites suitable for elimination (Lerapetritou et al., 2009). Here, we show that xenobiotic

metabolism genes of the cytochrome P450 family were markedly up-regulated in the regenerating

schistosome-infected liver following parasite elimination. These observations are thought to be

independent of the effect of PZQ administration, as PZQ-treated control mice did not show any

modulation in the transcriptional expression of genes associated with xenobiotic metabolism at the

time points we examined. Xenobiotics such as drugs and chemicals are mostly highly lipophilic

compounds that are difficult to excrete through the kidneys, and are able to cause injury to the liver

and other tissues. As the major site of xenobiotic metabolism, the liver produces hepatic enzymes

such as cytochrome P450 to remove these damaging compounds (Sturgill and Lambert, 1997). The

role of cytochrome P450 in active and recovering hepatic schistosomiasis is not clear at this stage

and will require future work to clearly elucidate its function. The expression of cytochrome P450

enzymes are in turn regulated by AhR, also known as the xenobiotic receptor, a ligand-dependent

transcription factor (Beischlag et al., 2008). It was reported previously that AhR-deficient mice

showed a decrease in the size of hepatocytes and the organ as a whole, indicating that AhR plays an

important role in the development of normal liver function (Fernandez-Salguero et al., 1995; Lahvis

et al., 2000). Consistent with the increased expression of xenobiotic genes, the AhR signalling

pathway was also shown to be up-regulated in our model of recovering liver, demonstrating the

significance of xenobiotic detoxification processes as a part of the regulation of hepatic function.

In summary, we present the most comprehensive known pathological and transcriptional

profile of the chronic and recovery phases of schistosome-infected livers in the murine model. We

show that granulomatous pathology and the extent of fibrosis were greatly reduced following

elimination of the adult parasite and the cessation of egg laying, and these events were accompanied

by a decrease in the numbers of immune cell infiltrates to the liver. Our findings indicate, to our

knowledge for the first time, the involvement of PPAR-α activation and the AhR signalling pathway

in the regenerative process of liver recovery. The significant up-regulation of metabolic genes in the

recovering liver as well marks the successful regression and remodelling of the liver.

Acknowledgements

Support of this work from the National Health and Medical Research Council (NHMRC) of

Australia is gratefully acknowledged (APP1080007). DPM is an NHMRC Senior Principal

Research Fellow. The authors would like to thank Mary Duke for the maintenance of the S.

japonicum life cycle, and the histology department at QIMR Berghofer, Australia. The author(s)

declare that they have no competing interests.

References

Anderson, S.P., Yoon, L., Richard, E.B., Dunn, C.S., Cattley, R.C., Corton, J.C., 2002. Delayed

liver regeneration in peroxisome proliferator-activated receptor-alpha-null mice. Hepatology 36, 544 - 554.

Aragon, A.D., Imani, R.A., Blackburn, V.R., Cupit, P.M., Melman, S.D., Goronga, T., Webb, T., Loker, E.S., Cunningham, C., 2009. Towards an understanding of the mechanism of action

of praziquantel. Mol Biochem Parasitol 164, 57 - 65.

Beischlag, T.V., Luis Morales, J., Hollingshead, B.D., Perdew, G.H., 2008. The aryl hydrocarbon

receptor complex and the control of gene expression. Crit Rev Eukaryot Gene Expr 18, 207

- 250.

Bonzo, J.A., Ferry, C.H., Matsubara, T., Kim, J.H., Gonzalez, F.J., 2012. Suppression of hepatocyte

proliferation by hepatocyte nuclear factor 4alpha in adult mice. J Biol Chem 287, 7345 -

7356.

Braissant, O., Foufelle, F., Scotto, C., Dauca, M., Wahli, W., 1996. Differential expression of

peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -

beta, and -gamma in the adult rat. Endocrinology 137, 354 - 366.

Braun, L., Mead, J.E., Panzica, M., Mikumo, R., Bell, G.I., Fausto, N., 1988. Transforming growth

factor beta mRNA increases during liver regeneration: a possible paracrine mechanism of

growth regulation. Proc Natl Acad Sci U S A 85, 1539 - 1543. Burke, M.L., Jones, M.K., Gobert, G.N., Li, Y.S., Ellis, M.K., McManus, D.P., 2009.

Immunopathogenesis of human schistosomiasis. Parasite Immunol 31, 163 - 176. Burke, M.L., McManus, D.P., Ramm, G.A., Duke, M., Li, Y., Jones, M.K., Gobert, G.N., 2010a.

Temporal expression of chemokines dictates the hepatic inflammatory infiltrate in a murine model of schistosomiasis. PLoS Negl Trop Dis 4, e598.

Burke, M.L., McManus, D.P., Ramm, G.A., Duke, M., Li, Y., Jones, M.K., Gobert, G.N., 2010b. Co-ordinated gene expression in the liver and spleen during Schistosoma japonicum

infection regulates cell migration. PLoS Negl Trop Dis 4, e686.

Burke, M.L., McGarvey, L., McSorley, H.J., Bielefeldt-Ohmann, H., McManus, D.P., Gobert, G.N.,

2011. Migrating Schistosoma japonicum schistosomula induce an innate immune response

and wound healing in the murine lung. Mol Immunol 49, 191 - 200.

Burri, L., Thoresen, G.H., Berge, R.K., 2010. The Role of PPARalpha Activation in Liver and

Muscle. PPAR Res 2010, 542359.

Chiaramonte, M.G., Mentink-Kane, M., Jacobson, B.A., Cheever, A.W., Whitters, M.J., Goad,

M.E., Wong, A., Collins, M., Donaldson, D.D., Grusby, M.J., Wynn, T.A., 2003. Regulation

and function of the interleukin 13 receptor alpha 2 during a T helper cell type 2-dominant

immune response. J Exp Med 197, 687 - 701.

Chuah, C., Jones, M.K., Burke, M.L., Owen, H.C., Anthony, B.J., McManus, D.P., Ramm, G.A.,

Gobert, G.N., 2013. Spatial and temporal transcriptomics of Schistosoma japonicum-

induced hepatic granuloma formation reveals novel roles for neutrophils. J Leukoc Biol 94, 353 - 365.

Chuah, C., Jones, M.K., Burke, M.L., McManus, D.P., Gobert, G.N., 2014a. Cellular and chemokine-mediated regulation in schistosome-induced hepatic pathology. Trends Parasitol

30, 141 - 150. Chuah, C., Jones, M.K., Burke, M.L., McManus, D.P., Owen, H.C., Gobert, G.N., 2014b. Defining

a pro-inflammatory neutrophil phenotype in response to schistosome eggs. Cell Microbiol 16, 1666 - 1677.

Crabb, D.W., Galli, A., Fischer, M., You, M., 2004. Molecular mechanisms of alcoholic fatty liver:

role of peroxisome proliferator-activated receptor alpha. Alcohol 34, 35 - 38.

Crosby, A., Jones, F.M., Kolosionek, E., Southwood, M., Purvis, I., Soon, E., Butrous, G., Dunne,

D.E., Morrell, N.W., 2011. Praziquantel reverses pulmonary hypertension and vascular

remodeling in murine schistosomiasis. Am J Respir Crit Care Med 184, 467 - 473.

de Oliveira, S., Reyes-Aldasoro, C.C., Candel, S., Renshaw, S.A., Mulero, V., Calado, A., 2013.

Cxcl8 (IL-8) mediates neutrophil recruitment and behavior in the zebrafish inflammatory response. J Immunol 190, 4349 - 4359.

Desvergne, B., Michalik, L., Wahli, W., 2004. Be fit or be sick: peroxisome proliferator-activated receptors are down the road. Mol Endocrinol 18, 1321 - 1332.

Dheda, K., Huggett, J.F., Bustin, S.A., Johnson, M.A., Rook, G., Zumla, A., 2004. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. BioTechniques 37,

112 - 114, 116, 118 - 119.

Doenhoff, M.J., Cioli, D., Utzinger, J., 2008. Praziquantel: mechanisms of action, resistance and

new derivatives for schistosomiasis. Curr Opin Infect Dis 21, 659 - 667.

el-Badrawy, N.M., Hassanein, H.I., Botros, S.S., Nagy, F.M., Abdallah, N.M., Herbage, D., 1988.

Effect of praziquantel on hepatic fibrosis in experimental Schistosomiasis mansoni. Exp Mol

Pathol 49, 151 - 160.

Erikstrup, C., Kallestrup, P., Zinyama-Gutsire, R.B., Gomo, E., van Dam, G.J., Deelder, A.M.,

Butterworth, A.E., Pedersen, B.K., Ostrowski, S.R., Gerstoft, J., Ullum, H., 2008.

Schistosomiasis and infection with human immunodeficiency virus 1 in rural Zimbabwe:

systemic inflammation during co-infection and after treatment for schistosomiasis. Am J

Trop Med Hyg 79, 331 - 337.

Everts, B., Perona-Wright, G., Smits, H.H., Hokke, C.H., van der Ham, A.J., Fitzsimmons, C.M.,

Doenhoff, M.J., van der Bosch, J., Mohrs, K., Haas, H., Mohrs, M., Yazdanbakhsh, M., Schramm, G., 2009. Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives

Th2 responses. J Exp Med 206, 1673 - 1680. Fernandez-Salguero, P., Pineau, T., Hilbert, D.M., McPhail, T., Lee, S.S., Kimura, S., Nebert,

D.W., Rudikoff, S., Ward, J.M., Gonzalez, F.J., 1995. Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268, 722 - 726.

Giannandrea, M., Parks, W.C., 2014. Diverse functions of matrix metalloproteinases during fibrosis. Dis Model Mech 7, 193-203.

Gobert, G.N., 2010. Applications for profiling the schistosome transcriptome. Trends Parasitol 26,

434-439.

Gobert, G.N., Nawaratna, S.K., Harvie, M., Ramm, G.A., McManus, D.P., 2015. An ex vivo model

for studying hepatic schistosomiasis and the effect of released protein from dying eggs.

PLoS Negl Trop Dis 9, e0003760.

Gryseels, B., Polman, K., Clerinx, J., Kestens, L., 2006. Human schistosomiasis. Lancet 368, 1106 -

1118.

He, Y.X., Chen, L., Ramaswamy, K., 2002. Schistosoma mansoni, S. haematobium, and S.

japonicum: early events associated with penetration and migration of schistosomula through

human skin. Exp Parasitol 102, 99 - 108.

Hesse, M., Piccirillo, C.A., Belkaid, Y., Prufer, J., Mentink-Kane, M., Leusink, M., Cheever, A.W.,

Shevach, E.M., Wynn, T.A., 2004. The pathogenesis of schistosomiasis is controlled by

cooperating IL-10-producing innate effector and regulatory T cells. J Immunol 172, 3157 - 3166.

Hines-Kay, J., Cupit, P.M., Sanchez, M.C., Rosenberg, G.H., Hanelt, B., Cunningham, C., 2012. Transcriptional analysis of Schistosoma mansoni treated with praziquantel in vitro. Mol

Biochem Parasitol 186, 87 - 94. Hol, J., Wilhelmsen, L., Haraldsen, G., 2010. The murine IL-8 homologues KC, MIP-2, and LIX

are found in endothelial cytoplasmic granules but not in Weibel-Palade bodies. J Leukoc Biol 87, 501 - 508.

Huang, Y.X., Xu, Y.L., Yu, C.X., Li, H.J., Yin, X.R., Wang, T.S., Wang, W., Liang, Y.S., 2011.

Effect of praziquantel prolonged administration on granuloma formation around

Schistosoma japonicum eggs in lung of sensitized mice. Parasitol Res 109, 1453 - 1459.

Issemann, I., Green, S., 1990. Activation of a member of the steroid hormone receptor superfamily

by peroxisome proliferators. Nature 347, 645 - 650.

Kasinathan, R.S., Sharma, L.K., Cunningham, C., Webb, T.R., Greenberg, R.M., 2014. Inhibition

or Knockdown of ABC Transporters Enhances Susceptibility of Adult and Juvenile Schistosomes to Praziquantel. PLoS Negl Trop Dis 8, e3265.

Kersten, S., Seydoux, J., Peters, J.M., Gonzalez, F.J., Desvergne, B., Wahli, W., 1999. Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting. J Clin Invest

103, 1489 - 1498. La Flamme, A.C., MacDonald, A.S., Huxtable, C.R., Carroll, M., Pearce, E.J., 2003. Lack of C3

affects Th2 response development and the sequelae of chemotherapy in schistosomiasis. J

Immunol 170, 470 - 476.

Lahvis, G.P., Lindell, S.L., Thomas, R.S., McCuskey, R.S., Murphy, C., Glover, E., Bentz, M.,

Southard, J., Bradfield, C.A., 2000. Portosystemic shunting and persistent fetal vascular

structures in aryl hydrocarbon receptor-deficient mice. Proc Natl Acad Sci U S A 97, 10442

- 10447.

Lerapetritou, M.G., Georgopoulos, P.G., Roth, C.M., Androulakis, L.P., 2009. Tissue-level

modeling of xenobiotic metabolism in liver: An emerging tool for enabling clinical

translational research. Clin Transl Sci 2, 228 - 237.

Modi, W.S., Yoshimura, T., 1999. Isolation of novel GRO genes and a phylogenetic analysis of the

CXC chemokine subfamily in mammals. Mol Biol Evol 16, 180 - 193.

Morey, J.S., Ryan, J.C., Van Dolah, F.M., 2006. Microarray validation: factors influencing

correlation between oligonucleotide microarrays and real-time PCR. Biol Proced Online 8, 175 - 193.

Okaya, T., Lentsch, A.B., 2004. Peroxisome proliferator-activated receptor-alpha regulates postischemic liver injury. Am J Physiol Gastrointest Liver Physiol 286, G606 - G612.

Pearce, E.J., MacDonald, A.S., 2002. The immunobiology of schistosomiasis. Nat Rev Immunol 2, 499 - 511.

Perry, C.R., Burke, M.L., Stenzel, D.J., McManus, D.P., Ramm, G.A., Gobert, G.N., 2011. Differential expression of chemokine and matrix re-modelling genes is associated with

contrasting schistosome-induced hepatopathology in murine models. PLoS Negl Trop Dis 5,

e1178.

Rakhshandehroo, M., Knoch, B., Muller, M., Kersten, S., 2010. Peroxisome proliferator-activated

receptor alpha target genes. PPAR Res 2010, 612089.

Ramadoss, P., Marcus, C., Perdew, G.H., 2005. Role of the aryl hydrocarbon receptor in drug

metabolism. Expert Opin Drug Metab Toxicol 1, 9 - 21.

Ribeiro-dos-Santos, G., Verjovski-Almeida, S., Leite, L.C., 2006. Schistosomiasis--a century

searching for chemotherapeutic drugs. Parasitol Res 99, 505 - 521.

Rodriguez-Vilarrupla, A., Lavina, B., Garcia-Caldero, H., Russo, L., Rosado, E., Roglans, N.,

Bosch, J., Garcia-Pagan, J.C., 2012. PPARalpha activation improves endothelial dysfunction

and reduces fibrosis and portal pressure in cirrhotic rats. J Hepatol 56, 1033 - 1039.

Rovai, L.E., Herschman, H.R., Smith, J.B., 1998. The murine neutrophil-chemoattractant

chemokines LIX, KC, and MIP-2 have distinct induction kinetics, tissue distributions, and tissue-specific sensitivities to glucocorticoid regulation in endotoxemia. J Leukoc Biol 64,

494 - 502. Silveira-Lemos, D., Fernandes Costa-Silva, M., Cardoso de Oliveira Silveira, A., Azevedo Batista,

M., Alves Oliveira-Fraga, L., Soares Silveira, A.M., Barbosa Alvarez, M.C., Martins-Filho, O.A., Gazzinelli, G., Correa-Oliveira, R., Teixeira-Carvalho, A., 2013. Cytokine Pattern of

T Lymphocytes in Acute Schistosomiasis mansoni Patients following Treated Praziquantel Therapy. J Parasitol Res 2013, 909134.

Singer, M., Sansonetti, P.J., 2004. IL-8 is a key chemokine regulating neutrophil recruitment in a

new mouse model of Shigella-induced colitis. J Immunol 173, 4197 - 4206.

Steinfelder, S., Andersen, J.F., Cannons, J.L., Feng, C.G., Joshi, M., Dwyer, D., Caspar, P.,

Schwartzberg, P.L., Sher, A., Jankovic, D., 2009. The major component in schistosome eggs

responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease

(omega-1). J Exp Med 206, 1681 - 1690. Sturgill, M.G., Lambert, G.H., 1997. Xenobiotic-induced hepatotoxicity: mechanisms of liver injury

and methods of monitoring hepatic function. Clin Chem 43, 1512 - 1526. Thenappan, A., Li, Y., Kitisin, K., Rashid, A., Shetty, K., Johnson, L., Mishra, L., 2010. Role of

transforming growth factor beta signaling and expansion of progenitor cells in regenerating liver. Hepatology 51, 1373 - 1382.

Thomas, P.G., Carter, M.R., Atochina, O., Da'Dara, A.A., Piskorska, D., McGuire, E., Harn, D.A.,

2003. Maturation of dendritic cell 2 phenotype by a helminth glycan uses a Toll-like

receptor 4-dependent mechanism. J Immunol 171, 5837 - 5841.

Vaillant, B., Chiaramonte, M.G., Cheever, A.W., Soloway, P.D., Wynn, T.A., 2001. Regulation of

hepatic fibrosis and extracellular matrix genes by the th response: new insight into the role

of tissue inhibitors of matrix metalloproteinases. J Immunol 167, 7017 - 7026.

van der Kleij, D., van den Biggelaar, A.H., Kruize, Y.C., Retra, K., Fillie, Y., Schmitz, M.,

Kremsner, P.G., Tielens, A.G., Yazdanbakhsh, M., 2004. Responses to Toll-like receptor

ligands in children living in areas where schistosome infections are endemic. J Infect Dis

189, 1044 - 1051.

van Liempt, E., van Vliet, S.J., Engering, A., Garcia Vallejo, J.J., Bank, C.M., Sanchez-Hernandez,

M., van Kooyk, Y., van Die, I., 2007. Schistosoma mansoni soluble egg antigens are

internalized by human dendritic cells through multiple C-type lectins and suppress TLR-induced dendritic cell activation. Mol Immunol 44, 2605 - 2615.

von Lichtenberg, F., Sher, A., McIntyre, S., 1977. A lung model of schistosome immunity in mice. Am J Pathol 87, 105 - 123.

Walesky, C., Edwards, G., Borude, P., Gunewardena, S., O'Neil, M., Yoo, B., Apte, U., 2013. Hepatocyte nuclear factor 4 alpha deletion promotes diethylnitrosamine-induced

hepatocellular carcinoma in rodents. Hepatology 57, 2480 - 2490. World Health Organization (WHO), 2015. Schistosomiasis: number of people treated worldwide in

2013. Wkly Epidemiol Rec 90, 25 - 32.

Wilson, M.S., Mentink-Kane, M.M., Pesce, J.T., Ramalingam, T.R., Thompson, R., Wynn, T.A.,

2007. Immunopathology of schistosomiasis. Immunol Cell Biol 85, 148 - 154.

Wynn, T.A., Thompson, R.W., Cheever, A.W., Mentink-Kane, M.M., 2004. Immunopathogenesis

of schistosomiasis. Immunol Rev 201, 156 - 167.

Xiao, S.H., Mei, J.Y., Jiao, P.Y., 2009. The in vitro effect of mefloquine and praziquantel against

juvenile and adult Schistosoma japonicum. Parasitol Res 106, 237 - 246.

Yang, Y.Q., Yang, H.Z., Zhang, C.W., 1984. Effects of pyquiton on pulmonary S. japonicum egg

granulomas in sensitized mice. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 6, 217 - 220.

You, H., Gobert, G.N., Duke, M.G., Zhang, W., Li, Y., Jones, M.K., McManus, D.P., 2012. The

insulin receptor is a transmission blocking veterinary vaccine target for zoonotic

Schistosoma japonicum. Int J Parasitol 42, 801 - 807.

You, H., McManus, D.P., Hu, W., Smout, M.J., Brindley, P.J., Gobert, G.N., 2013. Transcriptional responses of in vivo praziquantel exposure in schistosomes identifies a functional role for

calcium signalling pathway member CamKII. PLoS Pathog 9, e1003254. Zwingenberger, K., Richter, J., Vergetti, J.G., Feldmeier, H., 1990. Praziquantel in the treatment of

hepatosplenic schistosomiasis: biochemical disease markers indicate deceleration of fibrogenesis and diminution of portal flow obstruction. Trans R Soc Trop Med Hyg 84, 252

- 256.

Figure Legends

Fig. 1. Diagrammatic overview of infection and Praziquantel (PZQ) administration in a murine

model of hepatic schistosomiasis japonica. C57BL/6 mice were infected with 14 ± 1 Schistosoma

japonicum cercariae, and were first euthanised at 6 weeks p.i. before the commencement of PZQ

treatment to provide a baseline indication of the degree of fibrosis. Another group of infected mice

were administered an increasing dosage of PZQ (Group PI) over five consecutive days at 7 weeks

p.i. Uninfected mice were also treated with PZQ (Group PU) to assess the hepatotoxic effect of the

drug. Untreated infected (Group NI), PZQ-treated infected (Group PI) and the PZQ-treated

uninfected mice (Group PU) were euthanised at 10, 13, 14 weeks p.i. The NU group (not included

into this figure) represents the uninfected mice that did not receive PZQ treatment.

Fig. 2. Histopathological features of Schistosoma japonicum egg-induced hepatic granuloma in

C57BL/6 mice before and after Praziquantel (PZQ) treatment. Common features seen include:

lymphocytes and plasma cells (�), neutrophils (*), macrophages associated with fibroplasias (>),

(A) Uninfected liver (Group NU) shows normal tissue architecture, corresponding in age to week 6

mice. This histology is representative of uninfected mice throughout the time course (also Group

PU, PZQ-treated uninfected mice). (B - E) Normal disease progression Group NI (non-PZQ-treated

infected mice. (B) Schistosome eggs surrounded by a dense population of neutrophils, and rimmed

by macrophages within a band of proliferating fibrovascular tissue at 6 weeks p.i. (C) A dense

population of macrophages together with a small number of neutrophils present in the core of

granulomas surrounding the parasite eggs at 10 weeks p.i. A dense population of lymphocytes are

shown making up the outer rim (arrows). (D - E) Granuloma formed at 13 and 14 weeks p.i.

showing varying proportions of lymphocytes, fibrosis in a collagenous stroma and macrophages. A

variable population of neutrophils is scattered through this area, and most of the eggs present are

calcified. Granulomas in PZQ-treated at week 7 post cercarial challenge mice (Group PI) after (F) 3,

(G) 6 and (H) 7 weeks of PZQ treatment have started to decrease in size and show reduced cellular

infiltration, with only a scattering of lymphocytes and plasma cells, and a few macrophages present.

All liver sections shown are formalin-fixed, paraffin-embedded and stained with H&E. Scale bar =

100 µm.

Fig. 3. Parasitological and pathological comparisons between untreated Schistosoma japonicum-

infected (Group NI), Praziquantel (PZQ)-treated infected (Group PI) and PZQ-treated uninfected

mice (Group PU). (A) No worms were present in mice after PZQ treatment at all time points. (B)

There was no significant difference in the liver weight of the PZQ-treated (Group PI) and untreated

mice (Group NI) at any time-point. The degree of splenomegaly in the PZQ-treated mice (Group PI)

was reduced significantly following drug treatment. The weights of control mice (untreated,

uninfected) at week 6 are presented as a reference. (C) The number of egg clusters in the PZQ-

treated mice (Group PI) was greatly reduced after 3 and 7 weeks of PZQ treatment. (D) Hepatic egg

burdens did not differ significantly between PZQ-treated (Group PI) and untreated groups (Group

NI). Statistical significances between groups were determined using two-way ANOVA with Tukey

post hoc tests. Data are presented as mean ± S.E.M. (n = 6) * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001,

**** P ≤ 0.0001.

Fig. 4. Cellular infiltration in the murine liver before and after Praziquantel (PZQ) treatment.

Granuloma density (A – C; H&E stain) of the Schistosoma japonicum-infected, PZQ-treated mice

(Group PI) decreased significantly at all time points post-treatment. Collagen deposition (D – F;

Sirius red) in PZQ-treated livers (Group PI) was reduced significantly after 3 weeks of PZQ

treatment, and no significant differences were observed at either 6 or 7 weeks post-treatment,

compared with the livers from untreated infected mice (Group NI). Macrophage, neutrophil and

eosinophil infiltration (G – I, F4/80+; J – L, Leder stain (*); M – O, Giemsa stain) in the PZQ-

treated group livers (Group PI) decreased significantly post treatment. Scale bar = 100 µm. Control

mice (Group PU) were PZQ-treated uninfected mice.

Fig. 5. Quantitation of immunohistochemistry presented in Fig. 4. (A) Granuloma density was

determined by H&E staining, while specific cells were identified using the following stains. (B) The

degree of hepatic fibrosis was determined with Sirius red staining. (C) F4/80+ for macrophages. (D)

Leder stain for neutrophils. (E) Giemsa stain for eosinophils. Data are presented as mean ± S.E.M.

(n = 6) * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Fig. 6. Semi-quantification of immunolabelling during disease progression and recovery.

Hepatocyte replication activity as indicated by positive Ki67 stained nuclei (arrows), was on

average, (A) low in uninfected, (B) most frequent in livers from mice infected with Schistosoma

japonicum at 6 weeks p.i., (C) also frequent in chronically infected samples of mice 10 to 14 weeks

p.i. and (D) presented the lowest frequency in Praziquantel (PZQ)-treated animals. Tissue inhibitor

of metalloproteinase-1 (TIMP1) staining is characterised by a thin, intermittent band of

mononuclear cells with positive cytoplasmic staining around the periphery of granulomas in acutely

infected livers of mice 6 weeks p.i. (E) There was no significant TIMP1-positive staining in the

uninfected control samples. (F) During the acute phase of disease progression (6 weeks p.i.) a band

around the granuloma of positive cells is evident. (G) In chronically infected livers (10 to 14 weeks

p.i.), these cells form a more continuous and thicker band at this location. (H) In PZQ-treated livers,

the band of positively stained cells is again thin and intermittent. The open circles indicate S.

japonicum ova in the centre of granulomas. (A - D) Scale bar = 150 µm; (E - H) Scale bar = 360

µm.

Fig. 7. Quantification of immunolabelling during disease progression and recovery. The percentage

of cells positive for (A) Ki67 (a marker for cellular proliferation) and (B) tissue inhibitor of

metalloproteinase-1 (TIMP1) are presented for liver tissue of Praziquantel (PZQ)-treated uninfected

controls. Animals infected with Schistosoma japonicum and tissue collected at acute (6 weeks p.i.)

and chronic (10, 13 and 14 weeks p.i.) phases. Animals were infected with S. japonicum, treated

with PZQ at week 7 p.i. to clear the infection, and the tissue allowed to recover; tissues were

sampled at weeks 10, 13 and 14 p.i.. Significant differences between treatment groups (PI, PZQ-

treated infected mice; PU, PZQ-treated uninfected mice; NI, untreated infected mice) and weeks are

indicated by: * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001.

Supplementary Figure legend

Supplementary Fig. S1. Validation of microarray data of a subset of genes by quantitative real-

time PCR. Real-time PCR data are presented in the column graphs as copy number. Data are

presented as mean ± S.E.M. Genes are S100A9, S100 calcium binding protein A8; MPO,

Myeloperoxidase; Cytochrome P450 family (CYP2B9, CYP2A5, CYP8B1, CYP4A14); FMO,

Flavin containing Monooxygenase; INMT, Indolethylamine N-methyltransferase. Real-time PCR

was performed using three biological replicates, n = 3. Mouse groups were NU, untreated,

uninfected; NI, untreated, Schistosoma japonicum-infected; PI, Praziquantel (PZQ)-treated, S.

japonicum-infected; PU, PZQ-treated, uninfected. Significant differences between treatment groups

and weeks are indicated by: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Probe ID Definition

6 10 13 14

IL-8 Signaling

ILMN_2763245 CXCL1 Chemokine (C-X-C motif) ligand 1 24.19 18.41 20.02 23.64

ILMN_2925094/

2600421/2719256

ILMN_2711075 MMP9 Matrix metallopeptidase 9 2.88 7.03 2.86 13.23

ILMN_2654495 IQGAP1 IQ motif containing GTPase activating

protein 1

7.89 6.37 4.16 8.17

ILMN_1253972 IRAK3 IL-1 receptor-associated kinase 3 3.89 3.83 3.26 4.43

ILMN_2778655 VCAM1 Vascular cell adhesion molecule 1 6.81 3.75 3.26 4.2

ILMN_2473531/

3114641

ILMN_2953531 MYLC2B Myosin light chain, regulatory B 2.71 3.02 2.45 3

ILMN_2658804 RRAS Related RAS viral oncogene homolog 2.06 2.03 2.44 2.14

ILMN_1234223 PLD4 Phospholipase D family, member 4 3.83 2.73 2.4 2.25

ILMN_1215212 RHOB Ras homolog family member B 3.15 2.57 2.23 2.55

ILMN_2896601 ICAM1 Intercellular adhesion molecule 1 3.1 2.08 2.16 2.38

ILMN_2700715 CSTB Cystatin B (stefin B) 2.66 2.07 2.12 2.56

ILMN_2700848 ARRB2 Arrestin, beta 2 3.03 3.28 2.1 3.57

ILMN_2700166 CCND2 Cyclin D2 3.53 3.53 2.06 2.24

Table 1. Genes associated with IL-8 signalling that were up-regulated during the progression of Schistosoma

japonicum -infection in C57BL/6 mice at 6, 10, 13 and 14 weeks p.i., normalised to uninfected controls.

Gene

Symbol

2.43

Fold Change (weeks)

MPO Myeloperoxidase 12.97 16.29 4.67 23.52

PIK3R1 Phosphoinositide-3-kinase, regulatory

subunit 1

2.8 2.69 2.35

Table 2. Dendritic cell maturation-associated genes up-regulated during the progression of

Schistosoma japonicum infection in mice at 6, 10, 13 and 14 weeks p.i., normalised to

uninfected controls.

Probe ID Gene

Symbol

Definition Fold Change

(weeks)

6 10 13 14

Dendritic Cell

Maturation

ILMN_2631161 FCGR3A Fc fragment of IgG, low affinity IIIa,

receptor

14.8 13.4 9.4 9.5

ILMN_2748875 FCER1G Fc fragment of IgE, high affinity I,

receptor for; gamma polypeptide

7.54 5.53 3.9 5.6

ILMN_2777498 IL-1B IL-1, beta 5.53 3.75 3.2 4.3

ILMN_2867147 TYROBP TYRO protein tyrosine kinase binding

protein

4.16 3.26 2.6 3.9

ILMN_2473531/ PIK3R1 Phosphoinositide-3-kinase, regulatory

subunit 1

2.8 2.69 2.4 2.4

3114641

ILMN_2687403 FCGR2A Fc fragment of IgG, low affinity IIa,

receptor

3.95 2.67 2.3 3.3

ILMN_2733733 TLR2 Toll-like receptor 2 3.63 2.15 2.3 3

ILMN_2896601 ICAM1 Intercellular adhesion molecule 1 3.1 2.08 2.2 2.4

ILMN_3072427 IL1RN IL-1 receptor antagonist 7.97 3.25 2.1 3.8

�

� �

Table 3. Genes up-regulated in Schistosoma japonicum-infected mice after 3, 6 and 7 weeks

of Praziquantel (PZQ) treatment, each normalised to the corresponding time points for S.

japonicum-infected untreated animals.

Probe ID Gene

Symbol

Definition Fold (weeks)

3 6 7

Fatty Acid Metabolism

ILMN_2617625 Cyp2b9 Cytochrome P450, family 2, subfamily b, polypeptide 9

13.3 11.7 36.5

ILMN_2659997 Acaa1b Acetyl-Coenzyme A acyltransferase 1B 3.7 4.61 8.86

ILMN_2622671 Acsl1 Acyl-CoA synthetase long-chain family

member 1

3.01 2.91 4.44

ILMN_2925350 Aldh3a2 Aldehyde dehydrogenase family 3,

subfamily A2

3.53 4.58 5.32

ILMN_2645815 Cyp2a12 Cytochrome P450, family 2, subfamily a,

polypeptide 12

2.64 2.1 3.47

ILMN_2642975/ Cyp2d26 Cytochrome P450, family 2, subfamily d,

polypeptide 26

2.44 2.61 2.98

1227936

ILMN_2706120 Ehhadh 3-hydroxyacyl Coenzyme A dehydrogenase 2.14 2.27 3.76

ILMN_2648445 Slc27a2 Solute carrier family 27, member 2 3.18 2.24 3.67

Xenobiotic Metabolism

ILMN_2617625 Cyp2b9 Cytochrome P450, family 2, subfamily b,

polypeptide 9

13.3 11.7 36.5

ILMN_2925350 Aldh3a2 Aldehyde dehydrogenase family 3, subfamily A2

3.53 4.58 5.32

ILMN_2645815 Cyp2a12 Cytochrome P450, family 2, subfamily a,

polypeptide 12

2.64 2.1 3.47

ILMN_2769991 Cyp2c29 Cytochrome P450, family 2, subfamily c,

polypeptide 29

4.59 3.95 4.87

ILMN_2691059/ Cyp2c37 Cytochrome P450, family 2. subfamily c,

polypeptide 37

3.24 3.22 3.62

2691060

ILMN_2996640 Cyp2c50 Cytochrome P450, family 2, subfamily c,

polypeptide 50

3.57 3.12 3.67

ILMN_3074610 Cyp2c67 Cytochrome P450, family 2, subfamily c,

polypeptide 67

2.85 2.67 2.78

ILMN_2642975/ Cyp2d26 Cytochrome P450, family 2, subfamily d, polypeptide 26

2.44 2.61 2.98

1227936

ILMN_2753183 Cyp3a11 Cytochrome P450, family 3, subfamily a, polypeptide 11

3.73 2.44 3.66

ILMN_2928679 Cyp3a25 Cytochrome P450, family 3, subfamily a,

polypeptide 25

2.8 2.57 3.49

ILMN_2702903 Cyp2f2 Cytochrome P450, family 2, subfamily f,

polypeptide 2

5.94 4.13 11.6

ILMN_1234086 Dhdh Dihydrodiol dehydrogenase 2.47 2.02 2.93

ILMN_2838308 Fmo1 Flavin containing monooxygenase 1 2.18 2.2 3.48

ILMN_2680533 Gstt1 Glutathione S-transferase, theta 1 3.57 3.39 5.46

ILMN_2689880 Gstt2 Glutathione S-transferase, theta 2 2.97 3.59 4.82

ILMN_2665715 Gstt3 Glutathione S-transferase, theta 3 6.47 8.22 13.4

ILMN_2719069 Maob Monoamine oxidase B 3.16 2.11 4.89

ILMN_2641217 Sult1d1 Sulfotransferase family 1D, member 1 2.77 2.19 3.87

ILMN_2982764 Ugt2a3 UDP glucuronosyltransferase 2 family, polypeptide A3

2.59 2.39 4.16

ILMN_1228666 Ugt2b1 UDP glucuronosyltransferase 2 family,

polypeptide B1

3.55 2.71 4.43

ILMN_2930203 Ugt1a10 UDP glycosyltransferase 1 family,

polypeptide A10

2.55 2.22 2.79

�

Probe ID Gene

Symbol

Definition Fold

Change

ILMN_1222734 Aldh1a1 Aldehyde dehydrogenase family 1, subfamily A1 2.96

ILMN_1257020 Aldh1b1 Aldehyde dehydrogenase 1 family, member B1 3.83

ILMN_3100276 Aldh1l1 Aldehyde dehydrogenase 1 family, member L1 3.06

ILMN_2925350 Aldh3a2 Aldehyde dehydrogenase family 3, subfamily A2 5.32

ILMN_2874554 Aldh4a1 Aldehyde dehydrogenase 4 family, member A1 2.5

ILMN_1258158 Aldh6a1 Aldehyde dehydrogenase family 6, subfamily A1 2.13

ILMN_2775723 Aldh8a1 Aldehyde dehydrogenase 8 family, member A1 3.23

ILMN_2795106/2

739847

Cyp1a2 Cytochrome P450, family 1, subfamily a, polypeptide 2 2.98

ILMN_1248849 Gsta2 Glutathione S-transferase, alpha 2 2.37

ILMN_3138685 Gsta3 Glutathione S-transferase, alpha 3 2.34

ILMN_2892441 Gsta4 Glutathione S-transferase, alpha 4 3.58

ILMN_2792924 Gstk1 Glutathione S-transferase, kappa 1 2.41

ILMN_2862470 Gstm2 Glutathione S-transferase, mu 2 2.02

ILMN_1214964 Gstm4 Glutathione S-transferase, mu 4 2.43

ILMN_2633096/

1246321

ILMN_1254523 Gsto1 Glutathione S-transferase omega 1 2.95

ILMN_2680533 Gstt1 Glutathione S-transferase, theta 1 5.46

ILMN_2689880 Gstt2 Glutathione S-transferase, theta 2 4.82

Table 4. Ingenuity analysis showing the up-regulation of the pathway associated with aryl

hydrocarbon receptor signalling in Praziquantel (PZQ)-treated mouse livers compared with

untreated livers after 7 weeks of PZQ treatment.

Gstm6 Glutathione S-transferase, mu 6 2.55

Highlights

• Liver recovery after schistosomiasis involves both cellular and molecular components.

• Immune cell influx towards the liver granuloma is active during infection.

• PPAR-α is identified as a key mediator of the transcriptional events after cure.

• The aryl hydrocarbon receptor signalling pathway is central to detoxification after cure.