-
Immunobiology 215 (2010) 1011
Congenital exposure to Schistosoma mafuture immune response and
the disease
Ahmad A. Othmana,, Zeinab S. Shoheiba, Em
a
identication of activated hepatic stellate cells (HSCs) using
antibody against glial brillary acidic protein (GFAP).
et al. 2006). The principal events precipitating
chronicmorbidity during infection with Schistosoma mansoni(S.
mansoni) develop as a result of schistosome eggs that
ARTICLE IN PRESS
infection; PBS, phosphate buffered saline; RT-PCR, reverse
tran-
scription polymerase chain reaction; TGF-b, transforming
growthfactor beta; S. mansoni, Schistosoma mansoni.Corresponding
author at: Parasitology Department, Tanta Facultylodge in the
liver, gut, and other organs causinggranulomatous inammation that
ends in brosis and
0171-2985/$ - see front matter r 2009 Elsevier GmbH. All rights
reserved.
doi:10.1016/j.imbio.2009.04.004
of Medicine, Tanta, Egypt. Fax: +20 2 40 3407734.
E-mail address: [email protected] (A.A. Othman).In
conclusion, congenital exposure to S. mansoni seems to ameliorate
the immunopathological changes in futurepostnatal infections.r 2009
Elsevier GmbH. All rights reserved.
Keywords: Congenital exposure; Cytokines; GFAP; Hepatic stellate
cells; Immune response; Liver brosis; Schistosoma mansoni
Introduction
Schistosomiasis is a water-borne parasitic disease thatplagues
many tropical and subtropical regions all overthe world, aficting
over 200 million people (Gryseels
Abbreviations: BSA, bovine serum albumin; GFAP, glial
brillary
acidic protein; HRP, horseradish peroxidase; HSCs, hepatic
stellate
cells; IL-12, interleukin-12; MoAb, monoclonal antibody; p.i.,
post-bDepartment of Pathology, Faculty of Medicine, Tanta
University, Tanta, EgyptcDepartment of Medical Parasitology,
Faculty of Medicine, Suez Canal University, Ismailia, Egypt
Received 19 February 2009; received in revised form 28 March
2009; accepted 1 April 2009
Abstract
Schistosomiasis mansoni is a widespread parasitic infection that
may lead to several serious complications, such ashepatic
periportal brosis and portal hypertension, mainly due to deposition
of schistosome eggs in the tissues.However, people in endemic areas
infrequently exhibit severe pathology and complications; this may
be explained, inpart, by modulation of the disease in indigenous
populations by in utero exposure to the parasite. This
studyinvestigated the differences between mice born to Schistosoma
mansoni-infected mothers and those born to non-infected ones in
subsequent postnatal schistosomal infections. We found that the
intensity of infection, evidenced byhepatic egg load, was much
reduced in mice born to infected mothers. No difference was found
as regards total andSchistosoma-specic immunoglobulin levels except
for total IgG. The levels of gene expression of two
regulatorycytokines, namely interleukin-12 (IL-12) and transforming
growth factor beta (TGF-b) were found to be signicantlyincreased in
prenatally exposed animals. Moreover, liver brosis was signicantly
decreased in animals born to infectedmothers as revealed by
histopathological and histochemical examination as well as by
immunohistochemicalDepartment of Medical Parasitology, Faculty of
Medicine, Tanta University, Tanta, Egypt12
nsoni infection: Impact on theoutcome
an M. Saiedb, Rasha H. Solimanc
www.elsevier.de/imbio
-
mouse 3 weeks post-infection (p.i.). Age-matched
ARTICLE IN PRESSunobitissue damage. Although these egg-induced
granulomaseffectively sequester toxic egg products, they lead
tosevere hepatic brosis and portal hypertension (Wilsonet al.
2007).Clinical observations in schistosomiasis-endemic
areas show that major complications of Schistosomainfection
develop in approximately 412% of thepopulation, while the majority
of infected people remainasymptomatic or exhibit mild non-specic
symptoms(Tachon and Borojevic 1978). Also, indigenous peoplerarely
manifest acute or toxaemic symptoms, whereasindividuals from
non-endemic areas usually did (Caldaset al. 2008). One possible
reason for the presence orabsence of severe pathology is prenatal
exposure toschistosomiasis that modulates the future immuneresponse
later in life (Malhotra et al. 1997). This notionhas been suggested
by many authors and has beenexplored in experimental models (Camus
et al. 1976;Montesano et al. 1999; King 2001).Fibrosis is the
ultimate sequel of chronic hepatic
schistosomiasis. A characteristic type of periportalbrosis
occurs, which may lead to presinusoidal portalhypertension and
esophageal varices (Stavitsky 2004).Apparently, hepatic stellate
cells (HSCs) play a majorrole in the process of brous tissue
formation in theliver. They are responsible for synthesis of
componentsof extra-cellular matrix and several types of
collagen(Parola and Robino 2001). These cells reside in theDisses
spaces of the liver sinusoids, and they constitutea minor cell
type, roughly 58% of the total liver cells(Maubach et al. 2006).
Following chronic injury, HSCsdifferentiate into myobroblast-like
cells, acquiringcontractile and brogenic properties (Zhang et
al.2006). For immunohistochemical identication ofHSCs, traditional
antibodies against desmin (Yokoiet al. 1984), vimentin, and
a-smooth muscle actin(Baroni et al. 1996) were used. More recently,
glialbrillary acidic protein (GFAP), which is traditionallyused as
a marker for astrocytes of the brain, wasestablished as a marker
for HSCs (Lim et al. 2008).Fibrosis is a complex process that is
poorly understood,but several immunological and
non-immunologicaldeterminants are suggested, and the role of
cytokinesin this context is central (Alves-Oliveira et al.
2006).Cytokines play a crucial role in the evolution and
regulation of Schistosoma-induced immunopathology.In murine
models, immune responses to schistosomeantigens manifest a striking
shift from a moderate Th1to a robust Th2-dominated response.
Fibrosis and muchof the pathology is primarily mediated by Th2
while Th1responses are presumed to be protective (Reiman et
al.2006). However, recent evidence suggests that maintain-ing a
balanced and controlled Th1 or Th2 response iscritical in the case
of schistosomiasis for protectivegranuloma formation without
excessive pathology
A.A. Othman et al. / Imm102(Wilson et al. 2007). Transforming
growth factor betauninfected female mice were also mated with male
mice.After delivery, offspring were divided into two maingroups:
group I included 20 offspring born to non-infected mothers
(infected control group), and group IIincluded 20 offspring born to
S. mansoni-infectedmothers. Ten mice were left uninfected and
served asthe non-infected control for immunological parametersand
immunohistochemical staining (group N). Groups Iand II were then
infected subcutaneously withS. mansoni cercariae 60710
cercariae/animal. Miceof both groups were breast-fed, and were
8-week old atthe time of infection.At 12 weeks p.i., animals of
groups I, II, and N were
sacriced. Serum samples were collected from mice ofeach group
for detection of total and specic anti-(TGF-b) is secreted
ubiquitously by many cell types,including activated T cells and
activated macrophages(Miyazono et al. 1994). The bioactivity of
TGF-b isoften bidirectional, depending on target cells or
coex-isting mediators (Tsutsui and Kamiyama 1999). How-ever, its
role in Schistosoma-induced inammation andbrosis remains
controversial.The aim of the present study was to evaluate the
effect
of experimental congenital exposure to schistosomiasismansoni on
the subsequent postnatal infection. Weassessed the intensity of
infection, extent of brosis, aswell as several immunological
parameters in mice bornto Schistosoma-infected mothers in
comparison to thoseborn to non-infected ones.
Materials and methods
Parasite
Laboratory bred Biomphalaria alexandrina snails werepurchased
from the Schistosome Biological SupplyProgram, Theodore Bilharz
Research Institute (Giza,Egypt). After exposure to light for at
least 4 h,S. mansoni cercariae shed from the snails were used
toinfect the experimental animals of the study.
Animals and experimental design
Laboratory bred and parasite free Swiss albino mice(2025 g in
weight) were used in this study. Mice werepurchased from Theodore
Bilharz Research Institute(Giza, Egypt) and were housed and
infected inaccordance with the institutional guidelines. Eight
weeksold (n 10) female mice were infected with 200cercariae of S.
mansoni by subcutaneous injection asdescribed by Peters and Warren
(1969). Each of 10female mice was individually mated with a normal
male
ology 215 (2010) 101112Schistosoma antibodies. The liver of each
mouse was
-
put in the incubator at 37 1C for 6 h. Each test tube was
substrate. Sera giving absorbance values higher than
R35-72) rat MoAb. Following addition of the test
ARTICLE IN PRESSunobiolosamples and standards and washing,
HRP-conjugatedanti-mouse IgE (1 chain-specic, clone R35-118)
ratMoAb and biotin-labeled anti-mouse polyclonal IgGwere added to
each well. The reaction was readthe cut-off value ( mean absorbance
of wells withserum from control mice+3 SD) were consideredpositive.
Results were expressed in optical densities(ODs).
Estimation of total IgE and IgGCapture ELISA was used to
determine the levels of
total IgG and IgE in mouse sera based on protocolsdetailed by
the manufacturer (Pharmingen). Wells ofpolystyrene plates were
coated with antimurine poly-clonal IgG and antimurine IgE (1
chain-specic, cloneshaken then 0.1ml of the digest was examined
micro-scopically for counting S. mansoni eggs. The total eggcount
in 1 g liver tissue was then calculated (Cheever1968).
Evaluation of humoral factors
Estimation of S. mansoni-specic IgE and IgGSoluble antigens from
S. mansoni adult worms
(SAWA) were prepared as described (El Ridi et al.1993). In
indirect ELISA, wells of polystyrene plates(Costar, Cambridge, MA,
USA) were coated with500 ng soluble schistosome antigens, blocked
with 1%bovine serum albumin (BSA) in 0.05M phosphatebuffered saline
(PBS) pH 7.1, washed with PBS/0.05%Tween 20 (washing buffer), and
incubated with 100 mlserum diluted in washing buffer. Specic,
horseradishperoxidase (HRP)-labelled rat monoclonal antibody(MoAb)
to mouse IgG and IgE were purchased fromPharmingen (San Diego, CA,
USA), and used diluted1:1000 in washing buffer. HRP-labelled
streptavidin(Boehringer-Mannheim, Mannheim, Germany) wasused
diluted 1:5000. Reactivity was estimated spectro-photometrically at
492 nm after adding o-phenylenedia-mine (Sigma Chemical Co., St.
Louis, MO, USA)immediately removed and divided into three
portionsfor parasitological, immunological, histopathological,and
immunohistochemical studies.
Parasitological study
Liver egg load was estimated in groups I and II. Onegram from
each liver was weighed, and then put in a testtube containing 2ml
of 5% KOH and left overnight atroom temperature. The second day,
all test tubes were
A.A. Othman et al. / Immspectrophotometrically, and the serum
levels of IgGrecommended by the manufacturer.
Estimation of levels of IL-12 and TGF-b mRNAexpression by
semi-quantitative real-time PCR
Liver samples [10 randomly selected from groups Iand II, and 5
randomly selected from group N] weretaken and processed for RNA
extraction (MagNA Purecompact Nucleic Acid isolation kit I, Roche
Diagnos-tics, GmbH, Mannheim, Germany) according to
themanufacturers recommendations. First strand cDNAwas synthesized
from total RNA using TranscriptorFirst Strand cDNA Synthesis Kit
(Roche, Germany)according to the manufacturers
instructions.Determination of IL-12 and TGF-b mRNA expres-
sion by semi-quantitative reverse transcription polymer-ase
chain reaction (RT-PCR) was carried out usingb-globin as the
internal gene control. PCR was carriedout using Roche LightCycler
real-time PCR system.Briey, in a 1.5ml reaction tube on ice, the
followingcomponents were added in the same order they arementioned:
13 ml sterile H2O, PCR-grade, 2 ml Light-Cycler RT-PCR reaction mix
SYBR Green I, 1.6 mlMgCl2 stock solution (nal concentration 5mM), 2
mlcytokine primer mix (nal concentration 0.5 mM)and 0.4 ml
LightCycler RT-PCR enzyme mix, giving atotal volume of 19 ml. The
components were mixedgently then pipetted into pre-cooled
LightCycler capil-lary. One microliter of the cytokine RNA template
wasadded giving a nal volume of 20 ml. The primersequences for
IL-12 and TGF-b used in the study wereas follows:IL-12p40 (sense
50-GATGCTGGCCAGTACACC-30),
IL-12p40 (antisense 50-TCCAGCACGACCTCAATG-30),TGF-b (sense
50-CTACTACGCCAAGGAGGTC-30),TGF-b (antisense
50-TGACCCGCAGAGAGGCTA-T-30) (Techau et al. 2007). The optimal
conditions forPCR amplication were: an initial incubation at 95
1Cfor 30 s, 45 cycles of denaturation at 95 1C, annealing at55 1C
(10 s), and extension at 72 1C (13 s). The geneexpression for
measured cytokines and b-globin wasmeasured in copies/ml. For each
cytokine, the amountof RT-PCR product was normalized (as a ratio)
to thevalues obtained for b-globin, as an internal standard foreach
sample.
Histopathological, histochemical, andimmunohistochemical
study
All the studied specimens were xed in 10% formalinand
subsequently embedded in parafn, then submittedto
Haematoxylin and eosin (H&E) staining for histo-and IgE were
calculated from a standard curve as
gy 215 (2010) 101112 103pathological examination. The
composition of the
-
Statistical analysis
Data were presented as means7standard deviation.The probability
of signicant differences among dualmeans of groups was determined
by Students t-test.Fishers exact test was used for evaluating
GFAPexpression. Differences were considered non-signicantwhen
(P40.05), signicant (Po0.05), and highlysignicant (Po0.001). The
statistical analyses wereprocessed according to the conventional
procedures
increased in mice born to infected mothers (group II)
ARTICLE IN PRESSunobiology 215 (2010) 101112104with 0 indicating
the absence of positive staining; 1indicating the presence of mild
staining +; 2indicating the presence of moderate staining ++;3
indicating the presence of intense staining+++; and 4 indicating
the presence of verydistinct brownish cytoplasmic reaction to
GFAPwere considered positive. A semi-quantitative evalua-tion was
performed in order to estimate the numberof GFAP-positive HSCs
using scores from 0 to 4,granulomas was assessed and the granulomas
wereclassied into three types (brous, cellular, andbrocellular)
according to the predominant compo-nent (Costa-Silva et al.
2002).Massons trichrome staining to highlight broticchanges and to
conrm the type of granuloma. Theprocedure of Massons trichrome
stain was per-formed according to Jones (2002).Immunohistochemistry
was done for demonstrationof activated hepatic stellate cells using
antibodyagainst glial brillary acidic protein. Immunohisto-chemical
staining was performed on 35 mm sectionsfrom randomly selected 10
parafn blocks from eachof the infected groups and 5 blocks from
group N,using the UltraVision Detection System (Anti-Polyvalent,
HRP/DAB Ready-to-Use, Cat. #TP-015-HD, Lab Vision, USA). The
procedure of immunos-taining was conducted according to the
manufac-turers protocol. Briey, sections were deparafnizedwith
xylene and rehydrated with graded alcoholseries. Antigen retrieval
was done by immersing thesections in 10mmol/l citrate buffer (pH
6.0) for10min at 100 1C in microwave. Endogenous perox-idase
activity was blocked with hydrogen peroxideblock for 10min. After
thorough washing of thesections with phosphate buffered saline,
incubationwas done for 10min with Ultra V block to
preventnon-specic background staining, followed by rinsingthe
sections with PBS. Subsequently, an overnightincubation of the
sections with antibody againstGFAP (Ab-4 rabbit polyclonal
antibody, Cat. #RB-087-R7 Ready-to-Use, Lab Vision, USA) was doneat
room temperature in a humidity chamber. Thesections were then
washed with PBS and incubatedwith biotinylated goat anti-polyvalent
(secondaryantibody) for 10min at room temperature followedby
washing with PBS, then incubated with streptavi-din peroxidase
solution for 10min at room tempera-ture, then rinsed with PBS. The
reaction productswere visualized using
3-30-diamino-benzidine-tetra-hydrochloride (DAB), and the sections
were thencounterstained with Mayers haematoxylin, dehy-drated in
alcohol and mounted in DPX. Sectionsfrom the brain of a mouse were
used as positivecontrols, while negative controls were prepared
byomission of the primary antibody. Cells showing a
A.A. Othman et al. / Immintense staining ++++ (Gibelli et al.
2008).aPercent reduction was calculated according to the formula
[(group Icompared to mice born to non-infected mothers (group
I),and non-infected animals (group N), Po0.001.
Table 1. Liver egg count (mean7SD) recovered from miceborn to
non-infected mothers (group I) and mice born to
infected mothers (group II).
Group Egg count Reduction (%)a P-value
Group I (n 20) 46807191.3 Group II (n 20) 28307412.5 39.5
o0.001
meangroup II mean)/group I mean] 100.using Statistical Program
of Social Sciences (SPSS)software for windows, version 10.0.
Results
Hepatic egg load
Results of liver egg load are shown in Table 1. Therewas highly
signicant reduction in the mean numbers ofliver egg count in mice
born to infected mothers (groupII) compared to infected control
mice (group I). Themean liver egg count in group I at 12 weeks p.i.
was(46807191.3) compared to group II (28307412.5),Po0.001.
Antibody response
Results of antibody response 12 weeks p.i. aredemonstrated in
Fig. 1. There were no signicantdifferences between groups I and II
regarding totalIgE, Schistosoma-IgE and Schistosoma-IgG. On
theother hand, highly signicant increase in total IgG levelwas
observed in group II compared to group I,Po0.001.
Cytokine mRNA expression
Fig. 2 shows levels of mRNA expression of themeasured cytokines.
In the liver homogenates, levels ofIL-12 and TGF-b mRNA expression
were signicantlyPo0.001 (signicant reduction).
-
ARTICLE IN PRESSunobiTotal IgG
0
2
4
6
8
10
12
14
mg/
dl
Group NGroup IGroup II
Total IgE
3
4
5
/ml
Group NGroup IGroup II
A.A. Othman et al. / ImmHistopathological and histochemical
ndings
Histopathological examination revealed the presenceof
granulomatous reactions within the portal tracts.Each granuloma was
formed around a Schistosomaovum and was composed of cellular
collection ofeosinophils, histiocytes, lymphocytes, and plasma
cells.Peripheral broblasts with variable amounts of
collagendeposition were seen in brocellular and brous
types.Percentages of the different types of granulomas in
thestudied groups are illustrated in Fig. 3. In group I, thebrous
granulomas were the most prevalent type(Fig. 4A), whereas in group
II, the brocellular typepredominated (Fig. 4B). The brotic changes
within thegranulomas were highlighted by the Massons trichromestain
(Fig. 4C and D).
0
1
2IU
00.10.20.30.40.50.60.70.8
abso
rban
ce (4
92nm
) Group I Group II
Sch- IgG Sch- IgE
Fig. 1. Values of total IgG, total IgE, Schistosoma-IgG
(Sch-
IgG), and Schistosoma-IgE (Sch-IgE) in infected control mice
(group I) compared to mice born to infected mothers (group
II) expressed in means7SD. Signicant elevation in total IgGlevel
is observed in group II compared to group I (n
20).Po0.001.IL-12
00.5
11.5
22.5
33.5
4Group NGroup IGroup II
TGF-
1
2
3
4
5Group N
Group I
Group II
ology 215 (2010) 101112 105Immunohistochemical evaluation of
GFAPexpression
In the normal control group (group N), very fewscattered HSCs
showing faint positivity for GFAP weredetected. In both infected
groups, GFAP-positive HSCsappeared in two locations: (a) sinusoidal
(parenchymal)HSCs, which appeared as thin irregular positive
bandsalong the sinusoids with a dislocated nucleus and
(b)mesenchymal HSCs located in the hepatic lobule closeto the
portal tracts, within the portal tracts, and withinthe granulomas.
Immunohistochemical localization ofGFAP in both groups revealed
that group I showedhigher GFAP expression in comparison to group II
bothin sinusoidal (Fig. 5A and B) and mesenchymal (Fig. 5Cand D)
HSCs, indicating a more active and moreextensive brotic process in
group I compared to group II.GFAP expression in the infected groups
is illustrated inTable 2. The predominant grades of GFAP
expressionin group I were grades 3 and 4, while in group II
thepredominant grades were grades 1 and 2. The differencebetween
both groups was statistically signicant(Po0.05).
0
Fig. 2. Cytokine mRNA expression levels in liver tissues 12
weeks p.i. Vertical bars represent the mean (7SD) of
theseresults for each group (group N, n 5, groups I and II,n 10).
Signicant increase in levels of IL-12 and TGF-bmRNA was found in
mice born to infected mothers (group II),
Po0.001.
-
ARTICLE IN PRESS
0%
unobiGroup I (infected control group)
8.20%
29.4
A.A. Othman et al. / Imm106Discussion
Schistosomiasis remains a major cause of morbidity intropical
and subtropical countries (Engels et al. 2002). Itis quite
intriguing that in endemic areas few people
62.40%
Cellular fibrocellular fibrous
Fig. 3. Comparison between group I (infected control) and
group
different types of granulomas. Group I granulomas are mainly
of
predominate.
Fig. 4. Photomicrographs of schistosomal hepatic granulomas: (A)
a
most of them are of the brous variety arrows in infected
contro
prenatally exposed mice group II. The granulomas are brocell
granuloma in group I (Massons trichrome 400), and (D)
littletrichrome 400).Group II (born to infected mothers)
32.30%27.30%
ology 215 (2010) 101112develop severe pathology and sequelae,
while themajority show asymptomatic or mild pathology. In-tensity
and duration of the infection are majordeterminants, but other
factors are also involved. Theseinclude genetic background of the
host, nutritional
40.40%
Cellular fibrocellular fibrous
II (born to infected mothers) regarding the percentages of
the
the brous type while in group II brocellular granulomas
large number of granulomas formed around Schistosoma ova,
l group I (H&E 100), (B) fewer granulomas arrows inular
(H&E 100), (C) extensive brotic changes within thebrotic
changes within the granuloma in group II (Massons
-
ARTICLE IN PRESSunobiA.A. Othman et al. / Immstatus, parasite
strain differences, and frequency ofinfection. Maternal infection
status in that offspring ofthe infected mothers may be primed to
mount amodulated type of response at rst infection has beenproposed
by many authors in order to explain theindividual differences
(Butterworth and Thomas 1999).
Fig. 5. Hepatic immunohistochemical staining for GFAP: (A)
sect
expression located in sinusoidal HSCs arrow (immunoperoxidase
G
II showing grade 2 GFAP expression located in sinusoidal
HSCs
group I showing grade 3 GFAP expression located in both
mesenchy
400), and (D) section from group II showing grade 2
G(immunoperoxidase GFAP 400).
Table 2. GFAP expression in the infected groups.
Groups GFAP expression
Grade 0
GFAP staining
Grade 1
GFAP staining (+)
Gr
GF
N % N % N
Group I (n 10) 0 0 1 10 2Group II (n 10) 0 0 5 50 4
There is a signicant difference between both groups,
Po0.05.ology 215 (2010) 101112 107Epidemiology provides some
evidence: individuals whomove from non-endemic to endemic areas,
such asmigrants, travelers, or military personnel, often
expressmore severe acute and chronic schistosomiasis com-pared to
those who grew up within endemic areas (Nashet al. 1982).
ion from infected control group I showing grade 4 GFAP
FAP 400), (B) section from prenatally exposed mice grouparrow
(immunoperoxidase GFAP 400), (C) section frommal arrow and
sinusoidal HSCs (immunoperoxidase GFAP
FAP expression located in mesenchymal HSCs arrow
ade 2
AP staining (++)
Grade 3
GFAP staining
(+++)
Grade 4
GFAP staining
(++++)
% N % N %
20 4 40 3 30
40 1 10 0 0
-
ARTICLE IN PRESSunobiIn the present study, mice born to infected
mothersshowed signicant reduction in liver egg count com-pared to
infected control animals. Reduction in adultworm count may provide
an explanation for thesendings. Attallah et al. (2006) demonstrated
that adultworm count was signicantly reduced in the offspring
ofSchistosoma-infected mothers compared to mice born tonon-infected
ones, but no explanation was offered. Weassume that the situation
is perhaps similar to that ofconcomitant immunity in which
continuous antigenicstimulation (by the presence of some adult
worms)stimulates protective immune response against
furtherinfections. Early stimulation of the immune system by
inutero exposure to schistosomiasis may, therefore,provide quite
strong protective immunity against initialpostnatal infections. In
sum, congenital exposure toSchistosoma infection seems to reduce
the intensity ofinfection in subsequent postnatal exposure.As
regards the humoral immune response, no
signicant differences were noted in the levels
ofSchistosoma-specic IgG and IgE, as well as total IgEbetween mice
born to infected mothers and infectedcontrol animals. In contrast,
total IgG level wassignicantly increased in prenatally exposed
mice.Attallah et al. (2006) found that Schistosoma-specicIgG levels
were lower in prenatally exposed micecompared to control. However,
IgG levels were mea-sured earlier in the course of infection than
in our study.The increase in total IgG levels in our study should
beinterpreted with caution, for different classes ofIgG serve
different functions: whereas IgG2 class isconsidered to confer
immunity (Dunne et al. 1995;Noya et al. 1995), IgG4 are blocking
antibodiesthat may block IgE-mediated effects (Noya et al.
1995;King 2001), and may play a role in immunologicaltolerance in
prenatally sensitized individuals (Caldaset al. 2008). Although IgE
levels are correlated withresistance to infection especially after
treatment (Sattiet al. 1996), no difference was noted in our
studybetween both infected groups as regards total andspecic IgE
levels.Interleukin-12 (IL-12) is a heterodimer composed of
two disulde-linked polypeptide chains that are pro-ducts of two
distinct genes. It plays a pivotal role inpromoting cell-mediated
immune response. It promotesthe differentiation of uncommitted T
helper cells to theTh1 subset (Trinchieri and Scott 1999). A major
actionof IL-12 is its ability to induce the production of
IFN-g,doing so synergistically in combination with IL-2. Themajor
producers of IL-12 are the dendritic cells andmacrophages (Gately
et al. 1998).In our study, expression levels of IL-12 were
signicantly increased in mice born to infected motherscompared
to those born to non-infected ones. Few dataare available regarding
the role of IL-12 in schistoso-
A.A. Othman et al. / Imm108miasis, but all point out a benecial
effect (Wilson et al.2007). Vaccination of mice with parasite eggs
and IL-12inhibits the Th1 to Th2 shift and results in
ameliorationof hepatosplenic pathology following infection (Wynnet
al. 1995). Similarly, mice lacking IL-12 develop avigorous Th2
response that is detrimental during thechronic phase of infection
and display signicantmortality by 1215 weeks post-infection
(Hoffmannet al. 2000). The high levels of IL-12 may account in
partfor the ameliorated liver pathology and brosis noted inour
experiment.Transforming growth factor-b (TGF-b) is a pleio-
tropic cytokine that is involved in a number ofbiological
processes. It induces collagen and bronectinproduction by
broblasts. Further, it inhibits manyproducts of lymphocyte function
including T-cell pro-liferation, and plays a critical role in
T-cell homeostasis(Gorelik and Flavell 2000). It is produced by
specialsubset of CD4+ T helper cells, sometimes called Th3(Chatila
2005). Its role in Schistosoma-induced pathol-ogy and brosis is
unclear especially in humans. Someauthors believe that during
hepatic brogenesis, TGF-bhas a pivotal role in initiation and
progression ofdifferentiation of HSCs into myobroblasts
(Gressnerand Weiskirchen 2006; Chu et al. 2007). On the otherhand,
de Jesus et al. (2004) did not nd differences inTGF-b levels in
patients with different degrees ofhepatic brosis. Others suggested
that TGF-b is aregulatory cytokine which provides an effective
mechan-ism of control of the progression of brosis inassociation
with IL-10 (Kitani et al. 2003; Hesse et al.2004). Moreover, an
experimental study has demon-strated that brosis and much of the
pathology ofschistosomiasis is mediated by Th2 cytokines
especiallyIL-13, and not by TGF-b (Kaviratne et al. 2004).We found
that TGF-b mRNA expression levels were
signicantly higher in mice born to infected motherscompared to
infected control. This may account forreduced pathology and brosis
observed in our study,and this is probably related to its
inhibitory effect onT-cell proliferation. The brogenic activity of
TGF-bmight be offset by its immunoregulatory function.Furthermore,
IFN-g suppresses the brogenic activityof TGF-b by down-regulating
the expression of itsreceptors (Zhang et al. 2004). Thus, increased
IL-12 mayhave attenuated the brogenesis stimulated by
TGF-b.Presumably, other regulatory mechanisms may beinvolved, such
as CD4+CD25+ T regulatory cells,since these cells increase in
experimental Schistosoma-induced hepatic granulomas from 12% at 8
weeks to88% at 16 weeks during the course of infection, and
theyameliorate liver pathology in experimentally infectedmice
(Singh et al. 2005). However, this assumptionremains to be
tested.Fibrosis is the main culprit in Schistosoma-induced
pathology. The hepatic stellate cells (HSCs) play a
ology 215 (2010) 101112major role in this process, and there has
recently been
-
ARTICLE IN PRESSunobiunprecedented interest in this cell (and
its activatedform) as a prognostic indicator of progression of
liverbrosis, as well as a potential target for
therapeuticintervention (Moreira 2007). In addition,
multiplestudies have demonstrated a positive correlation be-tween
the severity of hepatic brosis and the number ofactivated stellate
cells present in the liver (Russo et al.2005). However, the
different stimuli that initiate andperpetuate HSC activation in
chronic liver diseases arepoorly understood. Remarkably, HSCs
affect adverselythe hepatic microcirculation. When activated,
theytransform into myobroblasts that contract aroundthe hepatic
sinusoids, increasing the vascular resistanceand contributing to
portal hypertension (Friedman2000).In our study, histopathological
and histochemical
examination revealed that most granulomas werebrocellular in
prenatally exposed offspring, whereasmost were brous in infected
control group. In addition,cellular granulomas were more abundant
in prenatallyexposed mice. Evident reduction in the activity of
HSCswas observed in mice born to infected motherscompared to
infected control group as demonstratedby immunohistochemical
staining for GFAP. Previousanimal studies did demonstrate
smaller-sized granulo-mas in offspring of infected mothers compared
tocontrol (Hang et al. 1974; Montesano et al. 1999;Attallah et al.
2006), but this is the rst time, to the bestof our knowledge, to
demonstrate the direct effect ofmaternal infection on HSC activity
in schistosomiasis.The reduced size of granulomas observed by
manyauthors and in our experiment (data not shown), and
thereduction in brosis in congenitally exposed mice maybe
attributed to attenuated Th2 response. The latter maybe due to, at
least in part, an increase in counter-regulatory cytokines such as
IL-12 and TGF-b.Furthermore, recent research stresses the role of
freeoxygen radicals as activators of HSCs (Loguercio andFederico
2003). As the overall liver inammation wasexpected to decrease in
congenitally exposed micedue to decreased egg deposition and
granuloma forma-tion, less free oxygen radicals would be produced
withattenuated stimulation of HSCs compared to infectedcontrol
mice.Perinatal immunological sensitization may occur by
transplacental and/or transmammary passage of schis-tosome
antigens (Carlier et al. 1980), anti-idiotypicantibodies (Montesano
et al. 1999), or immune com-plexes (Uhr et al. 1957). Attallah et
al. (2006) havealready detected Schistosoma antigens in the tissues
ofoffspring born to infected mothers particularly in theliver and
kidneys. In addition, several authors suggestedthat cellular
hypersensitivity to S. mansoni antigen couldin part be transmitted
in milk of infected mothers totheir infants (Eissa et al. 1989;
Santoro et al. 1977).
A.A. Othman et al. / ImmPresumably, exposure to parasite antigen
very early inthe development of the immune system results
inimmunological tolerance. The antigens can be no longerrecognized
as totally foreign. On initial postnatalexposure, the primed immune
system may be moreprone to develop less striking shift from Th1 to
Th2responses as seen in truly immunologically naveindividuals
(Caldas et al. 2008). A more balancedT-cell response may be
responsible for improvedimmunopathological processes and perhaps
better hostparasite interaction in indigenous populations.These
results can be relevant to humans since many
children are indeed born to infected mothers in endemicareas.
Unfortunately, no human studies have directlyshown that in utero
exposure to schistosomiasis affectsthe subsequent outcome of the
disease. However, vastamount of empirical evidence exists. For
example,children in Schistosoma-endemic areas rarely manifestacute
manifestations such as acute dermatitis orkatayama fever. These
children are born to motherswho had schistosomiasis during their
pregnancy, and areprobably already primed for many
anti-schistosomeresponses at the time of their rst exposure to
cercariae(Caldas et al. 2008). Interestingly, congenital
transmis-sion of Schistosoma japonicum to the offspring whenthe
mother acquires the infection during pregnancy was conrmed in pigs
(Willingham et al. 1999), and inhumans (Kikuchi 1957). If this
proves to be true forhuman schistosomiasis mansoni, this will add
to thecomplexity of the situation, and might provide astronger
background for immunomodulation in subse-quent infections.Not
surprisingly, the inuence of in utero exposure to
infections has been suggested in other chronic
parasiticdiseases. For example, in laria-endemic areas fetusesare
exposed to substantial amount of larial antigen.Exposure to
antigens at this stage of developmentpromotes tolerance by the
immune system (Hightoweret al. 1993). Thus, a child born to a
microlaremicmother is 2.9 times more likely to become
microlaremicthan a child born to an amicrolaremic mother.Likewise,
many individuals in endemic areas areasymptomatic microlaremics
(Kumar 1997). The samepattern is found in malaria-endemic areas
wherenon-endemic individuals react differently than indigen-ous
people (Rasheed et al. 1995). Moreover, transpla-cental transfer of
Opisthorchis felineus antigens in thehyperendemic foci of infection
does not prevent super-infection, but does prevent the acute phase
of diseaseand signicantly mitigates the organic lesions in
thechronic phase in spite of a very high intensity ofinfection
(Ozeretskovskaia 2000). One recent studydemonstrated that
experimental maternal infectionwith Opisthorchis viverrini affected
worm fecundityand worm load in acquired postnatal infections ofthe
offspring with the same parasite (Intapan and
ology 215 (2010) 101112 109Maleewong 2006).
-
109117.
Camus, D., Carlier, Y., Bina, J.C., Borojevic, R., Prata,
A.,
ARTICLE IN PRESSunobiCapron, A., 1976. Sensitization to
Schistosoma mansoni
antigen in uninfected children born to infected mothers.
J. Infect. Dis. 134, 405408.
Carlier, Y., Nzeyimana, H., Bout, D., Capron, A., 1980.
Evaluation of circulating antigens by a sandwich radio-
immunoassay, and of antibodies and immune complexes, in
Schistosoma mansoni infected African parturients and their
newborn children. Am. J. Trop. Med. Hyg. 29, 7481.
Chatila, T.A., 2005. Role of regulatory T cells in human
diseases. J. Allergy Clin. Immunol. 116, 949959.
Cheever, A.W., 1968. Conditions affecting the accuracy of
potassium hydroxide digestion in techniques for counting
S. mansoni eggs in tissues. Bull. World Health Organ. 39,
328331.
Chu, D., Luo, Q., Li, C., Gao, Y., Yu, L., Wei, W., Wu, Q.,In
conclusion, prenatal exposure to S. mansoniinfection affects the
immune response and the diseaseoutcome in the future postnatal
infections; the intensityof the infection as well as the
immunopathologicalchanges are moderated in congenitally exposed
hosts.These ndings warrant further investigations regardingthe
underlying mechanisms of immunomodulation hereand in other
parasitic infections, and they may also beapplicable to other
Th2-dominated diseases, such asallergy and bronchial asthma.
Finally, these results maygive us insight for prevention and
control strategies inschistosomiasis, and perhaps in other chronic
infectiousdiseases as well.
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ARTICLE IN PRESSA.A. Othman et al. / Immunobiology 215 (2010)
101112112
Congenital exposure to Schistosoma mansoni infection: Impact on
the future immune response and the disease
outcomeIntroductionMaterials and methodsParasiteAnimals and
experimental designParasitological studyEvaluation of humoral
factorsEstimation of S. mansoni-specific IgE and IgGEstimation of
total IgE and IgG
Estimation of levels of IL-12 and TGF-beta mRNA expression by
semi-quantitative real-time PCRHistopathological, histochemical,
and immunohistochemical studyStatistical analysis
ResultsHepatic egg loadAntibody responseCytokine mRNA
expressionHistopathological and histochemical
findingsImmunohistochemical evaluation of GFAP expression
DiscussionReferences