immune suppression in advanced chronic fascioliasis: an

1504 • JID 2007:195 (15 May) • Girones et al.

M A J O R A R T I C L E

Immune Suppression in Advanced ChronicFascioliasis: An Experimental Study in a Rat Model

Nuria Girones,1 M. Adela Valero,2 Maria A. Garcıa-Bodelon,2 Isabel Chico-Calero,1 Carmen Punzon,1 Manuel Fresno,1

and Santiago Mas-Coma2

1Centro de Biologıa Molecular “Severo Ochoa,” Departamento de Biologıa Molecular, Facultad de Ciencias, Universidad Autonoma de Madrid,Campus Cantoblanco, Madrid, and 2Departamento de Parasitologıa, Facultad de Farmacia, Universidad de Valencia, Burjassot-Valencia, Spain

Chronicity and Th2 immune responses are features of helminth infections in humans. The liver fluke promotesits own survival through several strategies to down-regulate the immune response of the host during the earlyphase of infection. However, there is no evidence that this modulation occurs much later. The immune responsein advanced chronic fascioliasis was analyzed in an experimental rat model at 20 weeks after infection. Cytokinequantification in infected rat serum revealed basal levels. The predominant immunoglobulin (Ig) isotype wasIgG1. Flow cytometry analysis of T cell (CD3+, CD4+, and CD8a+), B cell (CD45R+), and macrophage (CD11b+)populations in spleens showed no significant differences between infected and control rats. Mononuclear cellproliferation in the spleen in response to T and B mitogens was strongly inhibited in infected versus controlrats. During early chronic infection, there is a predominance of a Th2 response, which decreases in advancedchronic infection characterized by a persistent immune suppression.

Today, fascioliasis is considered to be an important hu-

man disease caused by 2 liver fluke species, Fasciola

hepatica and Fasciola gigantica (Fasciolidae), infecting

the liver of a wide range of mammals [1] that show a

marked variability in their immune response against

infection [2]. There are several geographic regions that

have been reported to be areas of hypoendemicity, me-

soendemicity, and hyperendemicity for fascioliasis in

Received 13 September 2006; accepted 9 December 2006; electronicallypublished 11 April 2007.

Potential conflicts of interest: none reported.Financial support: Spanish Ministry of Science and Technology (projects

BOS2002-01978 and SAF2006-09278); Red de Investigacion de Centros de En-fermedades Tropicales (project C03/04 of the Programme of Redes Tematicasde Investigacion Cooperativa) of the Fondo de Investigacion Sanitaria (FIS); andFIS, Spanish Ministry of Health (project PI030545). Immunological work was fundedby the Spanish Ministry of Education, Health, and Sport, the Ministry of Scienceand Technology, and Plan Nacional de Salud (projects SAF2002-03356 andSAF2004-0519), and Comunidad Autonoma de Madrid (08.3/0023.1/2001); by theRed Tematica Cooperativa de Investigacion en SIDA (RIS G03/173) and the RedTematica Recava (C03/01); and by Conselleria de Empresa, Universidad y Ciencia(projects 03/113, GV04B-125, and GVCOMP2006-106).

Reprints or correspondence: Dr. Manuel Fresno, Centro de Biologıa Molecular“Severo Ochoa,” Departamento de Biologıa Molecular, Facultad de Ciencias,Universidad Autonoma de Madrid, Campus Cantoblanco, 28049 Madrid, Spain([email protected]).

The Journal of Infectious Diseases 2007; 195:1504–12� 2007 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2007/19510-0016$15.00DOI: 10.1086/514822

humans, with prevalences and intensities ranging from

low to very high [3–7]. In zones of hyperendemicity,

the majority of adult human subjects are supposedly

experiencing the chronic phase of disease, which lasts

many years [4]. The liver fluke promotes its own sur-

vival through several strategies to down-regulate the

immune response of the host during the early phase of

infection. The liver fluke apparently secretes molecules,

known as excretory/secretory (E/S) products, that mod-

ulate or suppress host immune responses [8]. However,

to our knowledge, there is no evidence that modulation

of the immune response occurs much later (i.e., in

advanced chronic fascioliasis).

CD4+ T cells can be separated into 2 major subsets,

Th1 and Th2, on the basis of their cytokine secretion

patterns and function. Th1 cells produce many cyto-

kines, including interferon (IFN)–g and tumor necrosis

factor (TNF)–a, and promote the activation of mac-

rophages and the production of opsonizing antibodies.

Th1 cells mediate a delayed-type hypersensitivity re-

action and inflammatory responses. Th2 cells produce

many other cytokines, including interleukin (IL)–4 and

IL-10, and promote immediate-type hypersensitivity re-

actions involving IgE, eosinophils, and mast cells. Usu-

ally, the cytokines of each T cell subtype are mutually

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Immune Suppression in Chronic Fascioliasis • JID 2007:195 (15 May) • 1505

inhibitory for the differentiation and effector functions of the

reciprocal subset, resulting in the polarization of the immune

response to either type 1 or type 2 [9].

The Wistar rat model is a useful approach for pathological

research into the advanced chronic period of fascioliasis, be-

cause results can be extrapolated to chronic infection in hu-

mans. In the rat, fascioliasis is considered to be an advanced

chronic disease from 100 days after infection onward [10, 11].

Previous studies performed on T helper cytokines in rats ba-

sically concerned the early stages of fascioliasis, suggesting that

Fasciola species, similar to other helminth parasites, induce a

polarized Th2 response [12–14]. However, to our knowledge,

no studies have investigated the immune response of Fasciola

species in the advanced chronic stage in a rat model. Conse-

quently, we investigated cytokine, antibody, and spleen mono-

nuclear cell (Spm) proliferative responses in the advanced

chronic period in a Wistar rat model, by use of a parasite isolate

from an area where human fascioliasis is hyperendemic.

MATERIAL AND METHODS

Animals and experimental design. The study was approved

by the institutional committee on animal care at the University

of Valencia (Burjassot-Valencia, Spain). A liver fluke isolate was

obtained from the area of the Nile Delta region (in Egypt)

where human fascioliasis is hyperendemic (Behera Governor-

ate). Ten male Wistar rats (Iffa Credo) (body weight, 80–100

g) were housed in microisolator boxes and were maintained in

a pathogen-free room that was electrically heated and subject

to a light:darkness cycle of 12 h of light and 12 h of darkness

(i.e., conditions in compliance with the European Agreement

of Strasbourg [15]). A balanced commercial rodent diet (Panlab

Chow A04) and water were provided ad libitum. At the De-

partment of Parasitology at the University of Valencia, meta-

cercariae were obtained from experimentally infected Galba

truncatula snails and were stored in freshwater at 4�C until

required. Before being administered to the rats, the metacer-

cariae were checked for viability by use of the refractile ap-

pearance of the excretory granules as a criterion. G. truncatula

that shed the cercariae that gave rise to the metacercariae were

from a strain reared in a laboratory (in Heraeus-Votsch HPS

1500 and HPS 500 climatic chambers; the experimental con-

ditions were as follows: temperature, 20�C; photoperiod, 12 h

of light and 12 h of darkness; and relative humidity, 90%).

These snails were, in turn, monomiracidially infected [16]. Six

rats were infected with 20 metacercariae/rat, by use of an or-

ogastric syringe [17]. Infection was established by the detection

of eggs in feces. The number of eggs per gram of feces (epg)

shed by each rat was calculated using the Kato-Katz technique

(Helm-Test). Beginning 5 weeks after infection, fecal pellets

were collected, on a daily basis, directly from the cages of each

rat, and they were kept in a closed petri dish before exami-

nation, to avoid desiccation. The number of worms that suc-

cessfully developed in each rat was established by necropsy

performed using anesthesia (Fluothane; Zeneca Farma) fol-

lowed by exsanguination. Dissection was performed at 20 weeks

after infection. Worms specimens and bile from the common

bile duct were collected under a dissecting microscope in sterile

conditions; however, specimens from the rest of the organs

were also evaluated, especially the liver parenchyma.

Morphometric measurement techniques. The body size of

the liver fluke (expressed in millimeters squared) was measured

using a computer image analysis system (Optimas 5; Optimas),

with use of a color video camera (Sony DXC-930P) connected

to a stereomicroscope [18]. The total parasitic area was cal-

culated for each rat as the natural logarithm (with “ln” denoting

the logarithm having base e) of the sum of the body area of

all liver flukes present in the common bile duct (lnBA). For

bivariant correlations, the lnBA was used.

Blood sampling. For serum preparation, whole blood was

collected by means of venipuncture (in the great saphenous

vein) at 7, 8, 9, 10, 11, and 12 weeks after infection. At 20

weeks after infection, blood samples were obtained through

exsanguination. The blood was centrifuged at 760 ng at 4�C

for 10 min. Serum samples were stored at �80�C until analysis.

Blood eosinophils and leukocytes. Blood eosinophil and

leukocyte counts for each rat were calculated only after 20 weeks

after infection, by use of an automatic blood analyzer (Cell-

Dyn; Abbott Cientıfica). Verification of white blood cell dif-

ferentiation was performed through thin blood films stained

with Giemsa, and at least 100 cells were counted on each film.

Bile samples. The bile specimens were cultured aerobically

by inoculation onto a Mueller-Hinton plate and eosin-meth-

ylene blue agar, respectively. They were incubated at 37�C for

24 and 48 h, respectively.

Proliferation assays. Spleens from infected and control rats

were extracted, and cells were obtained using a 40-mm mesh

cell strainer (Becton Dickinson Labware). After red blood cells

were lysed with water for 5 s, the cells were washed twice in

Dulbecco’s modified Eagle medium (DMEM) and were resus-

pended in DMEM 10% fetal bovine serum (FBS). Cells were

plated in 96-well plates ( cells/200 mL) containing 5 mg/52 � 10

mL concanavalin A (ConA) and lipopolysaccharide (LPS), as

indicated. Proliferation was measured by incorporating 1 mCi

[3H]thymidine (Amersham Pharmacia Biotech) per well during

the last 24 h of a 3-day culture. Cells were then harvested on

a glass-fiber filter, by use of a Cell Harvester (Skatron Instru-

ments), and radioactivity was estimated.

Flow cytometry assays. A total of Spm cells per sample,510

obtained as described above, were washed in PBS containing

1% bovine serum albumin and 1% FBS. Cells were incubated

with antibodies to CD3+, CD4+, CD8a+, and CD11b+ coupled

to fluorescein isothiocyanate and CD3+, CD45R+, and CD11b+



Table 1. Kinetics of infection and morphometric parameters ob-served up to 20 weeks after infection in 6 rats with chronicFasciola infection.

Parameter Range (mean � SD)

PP, days 43–47 (43.8 � 1.6)

epg, by week after infection

7 144–968 (377.3 � 309.5)

10 1160–4264 (2457.3 � 1120.7)

14 1200–4128 (2360.0 � 1095.7)

Worms at 20 weeks after infection

Recovered per rat, no. 1–6 (3.3 � 1.8)

Recovered,a % 5–30 (16.6 � 9.3)

Body area,b mm2 270.5–719.7 (443.7 � 217.0)

NOTE. epg, no. of eggs per gram of feces; PP, prepatent period of eggemission.

a [(No. of flukes recovered/no. of cysts administered) � 100].b Sum of the body area of all liver flukes present in each rat bile duct.

Table 2. Leukocytes observed in the blood of 6 chronically infected rats at 20 weeks after infection and in 4 controlrats.

Rats Leukocytes, no. � 103 Neutrophils, % Lymphocytes, % Monocytes, % Eosinophils, %

Infected 1.1–4.7 (2.7 � 1.4) 12–25 (18.1 � 4.5) 62–74 (65.0 � 11.8) 4–23 (11.3 � 6.6) 3–6 (3.8 � 1.3)

Control 1.3–5.3 (2.9 � 1.6) 14–29 (20.4 � 6.3) 56–70 (65.4 � 5.6) 8–14 (9.8 � 2.5) 1–4 (2.8 � 1.3)

NOTE. Data are range (mean � SD).

coupled to phycoerythrin (BD Pharmingen) at a concentration

of 1 mg/ cells, in PBS, at 4�C for 20 min in the dark. Cells610

were washed 3 times and were resuspended in 500 mL of PBS.

Samples were analyzed in a FACSCalibur apparatus (BD

Biosciences).

Cytokine assays. Serum cytokine assays were performed

according to the directions of the manufacturers. Rat IL-4 Eli-

pair, Rat IFN-g Eli-pair, and Rat TNF-a Eli-pair were from

Diaclone Research. Rat IL-10 ELISA Set was from BD Biosci-

ences. Rat IL-1b ELISA Set was from R&D Systems.

IgG1, IgG2a, and IgE measurements in serum. Serum IgG

and IgE levels were determined using detergent-soluble F. he-

patica extract in ELISA. Mature flukes were removed from the

bile ducts of bovine livers. For the preparation of a detergent-

soluble extract, adult flukes were killed by freezing at �70�C

and were washed in PBS and drained. One gram of wet weight

of minced tissue was incubated in 10 mL of 50 mmol/L Tris

(pH 7.4), 150 mmol/L NaCl, 1% deoxycolate, 1 mg/mL of pro-

tease inhibitors (leupeptin, pepstatin, and aprotinin), and 1

mmol/L phenylmethylsulfonyl fluoride. The extract homoge-

nate was obtained by disruption with a polytron for 3 min on

ice, followed by centrifugation at 16,000g in a minifuge for 15

min. Quantification of protein in the resulting supernatant was

performed using the bicinchonic acid method. A total of 100

mg of protein extract per well of a 96-well plate was used.

Coating of the wells was performed in PBS at 4�C overnight.

Blocking was done at room temperature for 1 h, with the use

of PBS containing 0.2% Tween 20 and 3% low-fat dry milk.

Wells were washed 3 times with PBS containing 0.05% Tween

20 and 1% low-fat dry milk. Serum samples were diluted 1:50

in PBS and were incubated at room temperature for 1 h. Wells

were washed 3 times with PBS containing 0.05% Tween 20 and

1% low-fat dry milk. Antibodies against IgG1, IgG2a, and IgE

coupled to horseradish peroxidase (Serotec) were used at 1:

10,000 dilution and were incubated at room temperature for

1 h. Wells were washed 5 times with PBS containing 0.05%

Tween 20 and 1% low-fat dry milk, and they were developed

with phenylenediamine at room temperature for 30 min. Ab-

sorbance at 450 nm was measured in an EL 340 Biokinetics

microplate reader (Biotek Instruments).

Statistical analyses. Cytokines, antibody responses, prolif-

eration indexes, eosinophils, and leukocytes were compared

using the nonparametric Mann-Whitney U test. All experi-

ments were repeated 2 or more times, with similar results

achieved in each case. Bivariant correlation (Spearman corre-

lation) was used for the analysis of cytokine levels (TFN-a,

IFN-g, IL-10, IL-4, and IL-1b), immunoglobulin subclasses

(IgG1, IgG2a, and IgE), epg, number of parasites, and lnBA.

was considered to be statistically significant.P ! .05

RESULTS

Development of F. hepatica chronic infection in rats. The

results of the experimental infections are summarized in table

1. All infected rats were parasitized (mean no. of parasites, 3.3

worms/rat). The average number of eggs emitted daily increased

progressively until 78 days after infection, and, afterward, a

steady decrease in the shedding of eggs was observed until 95

days after infection, when the coprological study was com-

pleted. Significant differences in the average of the total values

of leukocytes and percentages of neutrophils, lymphocytes,

monocytes, and eosinophils were not detected between infected

rats and control rats at 20 weeks after infection (table 2). Results

of all microbiological analyses of the bile samples obtained from

infected rats and control rats were negative.

Immunoglobulin production during infection. Specific

IgG and IgE responses were induced in all infected rats (figure

1). A predominance of the IgG1 versus the IgG2a subclass was

observed in serum samples from 7 to 12 weeks after infection.



Figure 1. Effect of liver fluke infection on immunoglobulin (Ig) E, IgG1,and IgG2a levels in infected rats ( ) versus control rats ( ). Then p 6 n p 4bars denote SEs. OD, optical density.

Figure 2. Correlation between IgG1 and the number of eggs per gramof feces (epg) shed between 7–12 weeks after infection ( ). Then p 36bars denote SEs. For a proper representation, epg denotes values dividedby 10,000. Significant correlations were obtained between IgG1 levelsand epg ( ). was considered to be statistically significant.r p 0.460 P ! .05OD, optical density.

A significant increase in IgG1 and IgG2a levels in the serum

of infected rats, with respect to control rats, was detected. How-

ever, to a lesser extent, the IgE levels of infected rats had also

significantly increased from 7 to 12 weeks after infection, in

comparison with levels in serum samples from control rats.

The bivariant correlation of IgG1, IgG2a, and IgE levels in each

infected rat at 7, 8, 9, 10, 11, and 12 weeks after infection and

the weekly average of egg production (i.e., the epg) at 7, 8, 9,

10, 11, and 12 weeks after infection was calculated (figure 2).

A significant negative correlation was obtained between IgG1

levels and the epg ( ; ) (figure 2). Significantr p �0.442 P ! .05

correlations were detected between the percentage of eosino-

phils and IgG2a ( ; ) and between lnBA andr p 0.724 P ! .05

IgG1 ( ; ).r p 0.886 P ! .05

Cytokine production during infection. At 7, 10, and 20

weeks after infection, cytokine quantification in serum samples

from infected rats showed a predominance of Th2 cytokines

versus Th1 cytokines, compared with quantification in serum

samples from control rats (figure 3). No significant differences

in the secretion of type 1 cytokines IFN-g and TNF-a were

detected between infected and control rats at any time points

tested. Nevertheless, an increase in TNF-a production was ob-

served among the infected rats (figure 3). IL-1b, IL-10, and IL-

4 levels at 7 weeks after infection were significantly higher in

infected rats than in control rats. IL-10 and IL-4 levels decreased

at 10 and 20 weeks after infection. No significant differences

were detected in the levels of secretion of IL-1b, IL-10, and IL-

4 at 20 weeks between infected and control rats. Nor were any

significant correlations found between the number of parasites

and the TNF-a, IFN-g, IL-10, IL-4, and IL-1b levels in each

infected rat at 7, 10, and 20 weeks after infection. However,

analysis of the association between the TNF-a, IFN-g, IL-10,

IL-4, and IL-1b levels in each infected rat at 20 weeks after

infection and the parasitic area total of all parasites in the

common bile duct (lnBA) revealed a significant negative cor-

relation between lnBA and the TNF-a level ( ;r p �0.845 P !

). A significant negative correlation was observed between.05

the IL-4 level and the average egg production (the epg) at 7

and 10 weeks after infection ( ; ).r p �0.765 P ! .05

Spleen cell proliferative responses. Flow cytometry analysis

of T cell (CD3+, CD4+, and CD8a+), B cell (CD45R+), and

macrophage (CD11b+) populations in spleens showed no sig-

nificant differences between infected and control rats at 20

weeks after infection (figure 4). In experiments performed to

evaluate the proliferative response to mitogens (which are con-

sidered to be nonspecific) during the advanced chronic period

of the disease, Fasciola-infected Spm cell ConA activation from

parasitized rats showed a significant decrease in proliferation,

compared with Spm cells of healthy rats ( ). When theP ! .05

cells were stimulated with LPS, a significant decrease in the

proliferation by Spm cells of parasitized rats, compared with

Spm cells of healthy rats, was also found ( ) (figure 5).P ! .05

In conclusion, a pronounced suppression of mitogen-induced

proliferative response was observed. This immune suppression

was detected in all parasitized rats, independent of the worm

burden (1–6 liver flukes/rat)—that is, suppression can be gen-

erated by a single adult parasite.

DISCUSSION

The present study describes the immune response of fascioliasis

in a rat model, showing that, in early chronic infection, there



Figure 3. Effect of liver fluke infection on serum cytokine production (interferon [IFN]-g, tumor necrosis factor [TNF]–a, interleukin [IL]–4, IL-10, andIL-1b) in infected rats ( ) at 7, 10, and 20 weeks after infection vs. control rats ( ). Gray circles denote control rats, white circles denoten p 6 n p 4infected rats, and black circles denote average values for infected rats. **Statistically significant differences. was considered to be statisticallyP ! .05significant.

is an increase in type 2 cytokines that changes to a decrease

toward the advanced chronic phase. This finding correlates with

a predominance of IgG1-type immunoglobulins specific for

Fasciola species antigens at different times after infection, which

is characteristic of a type 2 response.

At 20 weeks after infection, Spm cells showed no statistical

differences in lymphoid and myeloid spleen cell populations,

nor in total leukocytes and the percentage of eosinophils. These

cells were unresponsive to mitogens, compared with the Spm

cells of control rats. This finding is indicative of a persistent

immune suppression. All the above findings are in agreement

with the findings of other studies of different host species that

have included infected rats but that have been performed at

earlier stages of infection [19–26]. The reason for this unre-

sponsiveness is not yet known. The E/S Fasciola products and/

or the presence of regulatory T (TR) cells are possibly respon-

sible for this effect; therefore, further research needs to be done.

This is the first time that immune suppression has been de-

scribed in advanced chronic fascioliasis in a rat model.

Blood eosinophils and leukocytes. Blood eosinophilia and

leukocytosis have been described in different host species in

fascioliasis [2, 27]. Eosinophils are involved in the antibody-

dependent cell-mediated cytotoxicity and participate in the an-

tibody-mediated destruction of juvenile forms of F. hepatica in

a variety of host species [28]. In experimental fascioliasis in

rats, eosinophilia has been described, reaching a maximum

value in the acute phase (4 weeks after infection) and exhibiting

a gradual decrease to 10 weeks after infection [29]. The data



Figure 4. Flow cytometry analysis of spleen mononuclear cells isolatedat 20 weeks after liver fluke infection. There were no significant statisticaldifferences in cell surface markers for T cells under basal, concanavalinA (ConA) stimulation, and lipopolysaccharide (LPS) stimulation conditions.For both infected and control rats, black bars denote CD3+ T cells; darkgray bars, CD4+ T cells; medium gray bars, CD8a+ T cells; light gray bars,CD45R+ B cells; and white bars, CD11b+ macrophages. The bars denoteSEs. was considered to be statistically significant.P ! .05

Figure 5. Effect of liver fluke infection on spleen cell proliferation inresponse to mitogens at 20 weeks after infection. White boxes denotecontrol rats, and black boxes denote infected rats. Significant inhibitionsof the proliferation index were observed in cultures stimulated withconcanavalin A (ConA) and lipopolysaccharide (LPS). The bars denote SEs.**Statistically significant differences. was considered to be sta-P ! .05tistically significant.

obtained in the present study of advanced chronic fascioliasis

show that, although in some rats, the rate of eosinophils is

higher than that in the control rats, the average is only slightly

higher, and significant differences were not detected. As in other

helminth infections, eosinophilia is one of the hallmarks of

human fascioliasis [30] and is probably related to the early

diagnosis of the disease in developed countries (in the acute

or early chronic phase), or it is likely that most of the parasites

eventually become trapped in the liver parenchyma [4]. Reports

of infections in areas where human fascioliasis is endemic show

that eosinophilia is not always present and, in some cases, is a

useless indicator of infection (e.g., in Peru, only 47% of patients

with chronic cases show eosinophilia [31], which is likely to

result from the delayed diagnosis in areas of hyperendemicity,

where the parasite usually reaches the bile duct [4]).

Immunoglobulin subclasses. A predominant Th2 cytokine

response, along with antifluke IgG1 in serum, was detected in

the present study, which agrees with the findings of other in-

vestigators [32]. Antibody responses in rats in the acute and

chronic phase (1–21 weeks) of disease show a marked pre-

dominance of IgG1 over IgG2a isotypes. During the first weeks

after infection, IgG1 quickly increases, whereas IgG2a slowly

increases and reaches the highest values at 5–7 weeks after

infection [32]. Humans infected with F. hepatica develop spe-

cific antibodies of the IgM, IgA, IgE, and IgG class. The im-

munoglobulin responses of liver fluke–infected humans to E/

S antigens and to a fluke cysteine proteinase, cathepsin L1,

showed that the predominant isotypes elicited by infection were

IgG1 and IgG4 [33]. An association between egg counts and

specific IgG antibody levels exists in humans [34].

Cytokines. Previous studies of Th cytokines in rats con-

cerned only the early stages of fascioliasis. During the first 2

weeks of infection, F. hepatica induced a transient Th0 cytokine

profile, followed by down-regulation of the cellular response

and the induction of a Th2 cytokine profile [12, 13]. In infected

rats, the presence of higher levels of IL-10 and IL-4 at 7 weeks



after infection and of IL-10 at 10 weeks after infection was

observed, findings that agree with previous observations re-

garding early infection. These findings suggest that the induc-

tion of IL-10 and IL-4 may suppress IFN-g production by Th1

cells and inhibit activation of macrophages. Nevertheless, cy-

tokine production is absent in the advanced chronic phase. IL-

4 limits the fecundity and survival of other helminths [35].

Likewise, we demonstrated that there is a negative correlation

between the liver fluke epg and IL-4, at least until 10 weeks

after infection. In some of the infected rats, TNF-a also in-

creased at 10 weeks after infection, which does not correspond

to a typical Th2 response. TNF-a is implicated in the regulation

of Th2 responses in other helminth infections, apparently reg-

ulating worm expulsion [36]. Unfortunately, the role of TNF-

a during liver fluke infection still remains unknown. IFN-g

and IL-4 play an important role in the establishment of chronic

helminth infection, parasite development, and worm expulsion

[37]. Moreover, IL-4 and IL-10 can act synergistically, inhibiting

the production of reactive nitrogen oxides, which up-regulate

IL-12 production and inflammatory responses [38].

Lymphocyte proliferative responses. To date, all mecha-

nisms described merely explain the advantage of the parasite

in the phases of rapid parasite growth within the liver paren-

chyma and establishment within the bile ducts—that is, as a

mechanism to avoid an immune response during the first stages

of liver penetration [22, 39, 40]. A parasite-induced immune

suppression in advanced chronic fascioliasis has been dem-

onstrated for the first time, which might correlate with the

immunomodulatory effects of E/S products released by the

adult fluke observed in vitro, in the absence of cytokine secre-

tion. High doses of F. hepatica and F. gigantica E/S products

released by the adult inhibit lymphoproliferation induced by

ConA in lymphocytes from various animal species [18, 23, 25,

26]. The liver fluke molecules that may induce immune sup-

pression have yet to be characterized, but they most likely are

actively secreted by the parasite. Candidate immunosuppressive

molecules include glycoconjugates sloughed from the parasite

surface glycocalyx, phosphorylchloline-rich antigens [41], Ku-

nitz-type serine proteinase inhibitors [42], cysteine proteinases

[43], and cathepsin L proteases [44]. On the other hand, the

role of TR cells in chronic helminth infections has been de-

scribed elsewhere [45]. TR cells are characterized by the pre-

dominant production of IL-10 and/or transforming growth fac-

tor b (TGF-b). In our study, IL-10 was not induced in the

advanced chronic phase, but other regulatory cytokines were

not tested. To our knowledge, there are no studies of TGF-b

during advanced chronic fascioliasis. Therefore, the role of TR

cells cannot be excluded in the maintenance of immune sup-

pression. Future experiments will address this hypothesis. The

present study proves that immune response modulation occurs

in advanced chronic fascioliasis. The proliferative response of

Spm cells to ConA and LPS significantly decreased at 20 weeks

after infection, which is in agreement with immune suppression

probably caused by an unknown fluke E/S product. This phe-

nomenon has also been detected in schistosomiasis. Many stud-

ies clearly demonstrate that the chronic phase of schistosomiasis

is characterized by a state of immune hyporesponsiveness ex-

hibited as a reduced ability of host cells to proliferate [46].

Chronicity, immune suppression, and Th2-type immune re-

sponses are characteristic features of human infection with mul-

ticellular parasites [47]. Immune suppression and Th2 re-

sponses have been attributed to chronic helminthic infection.

In both laboratory animals and ruminants, fascioliasis, like

other helminth infections, is a potent inducer of Th2 responses

that impair the ability to mount any effective Th1 responses

against bacteria and other pathogens [48, 49]. A high risk of

bacterobilia in advanced experimental chronic fascioliasis has

been described in a rat model and supports this fact [50].

Extrapolation of the results obtained in the rat model to human

infection gives a new dimension to chronic fascioliasis. The

effect of advanced chronic fascioliasis on the immune system,

particularly parasite-induced immune suppression, and the ef-

fect of cytokines controlling polarization of the Th1 or Th2

arms pose a great risk for individuals to acquire concomitant

infections (with viruses, bacteria, protozoa, and other hel-

minths). This observation of coinfections in humans is often

found in areas where fascioliasis is endemic (e.g., the Nile Delta

region in Egypt, which is where the fluke isolate used in the

present study originated [51]).

Acknowledgments

We thank M. D. Bargues and M. Kouhbane (Valencia, Spain) for col-lecting and rearing lymnaeid snails; for liver fluke infection experimentsof snails; and for obtaining, keeping, and providing the metacercariae used.We also thank M. V. Periago (Valencia, Spain) for her collaboration inexperimental rat studies. We also acknowledge the facilities provided byM. Mostafa, Z. Youssef, and Y. Abd El-Wahab (Egyptian Ministry of Healthand Population, Cairo, Egypt), the Behera Survey Team (Damanhour,Egypt), and F. Curtale and P. Barduagni (Italian Cooperation, ItalianEmbassy, Cairo, Egypt). We thank the Servicio de Analisis Clıni-cos del Hospital Militar de Valencia (Spain) for the hematologicaldeterminations.

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