Immunobiology 214 (2009) 712–727 Immunomodulatory effects of curcumin treatment on murine schistosomiasis mansoni Gamal Allam a,b, a Department of Zoology, Faculty of Science, Beni-Sueif University, Beni-Sueif, Egypt b College of Medicine and Medical Sciences (CMMS), Taif University, Taif, Saudi Arabia Received 7 October 2008; received in revised form 28 November 2008; accepted 29 November 2008 Abstract Curcumin is a polyphenol derived from the dietary spice turmeric. It has been shown to regulate numerous transcription factors, cytokines, adhesion molecules, and enzymes that have been linked to inflammation. In addition to inhibiting the growth of a variety of pathogens, curcumin has been shown to have nematocidal activity. The present study was designed to evaluate the schistosomicidal activity of curcumin in vivo as well as immunomodulation of granulomatous inflammation and liver pathology in acute schistosomiasis mansoni. Mice were infected each with 80 Schistosoma (S.) mansoni cercariae and injected intraperitoneally with curcumin at a total dose of 400 mg/kg body weight. Curcumin was effective in reducing worm and tissue-egg burdens, hepatic granuloma volume and liver collagen content by 44.4%, 30.9%, 79%, and 38.6%, respectively. Curcumin treatment restored hepatic enzymes activities to the normal levels and enhanced catalase activity in the liver tissue of infected mice. Moreover, hepato-spleenomegaly and eosinophilia induced by S. mansoni infection were largely improved with curcumin treatment. Infected mice treated with curcumin showed low serum level of both interleukin (IL)-12 and tumor necrosis factor alpha (TNF-a), but IL-10 level was not significantly altered. Specific IgG and IgG1 responses against both soluble worm antigen (SWAP) and soluble egg antigen (SEA) were augmented with curcumin treatment, but IgM and IgG2a responses were not significantly changed. In conclusion, curcumin treatment modulates cellular and humoral immune responses of infected mice and lead to a significant reduction of parasite burden and liver pathology in acute murine schistosomiasis mansoni. r 2008 Elsevier GmbH. All rights reserved. Keywords: Curcumin; Schistosoma mansoni; Immunomodulation; Cytokine; Antibody Introduction Schistosomiasis is one of the most prevalent parasitic infections worldwide. An estimated 779 million people are at risk for schistosomiasis, with 207 million infected in 76 countries and territories (Steinmann et al., 2006; Lammie et al., 2006). There are three main schistosome species causing schistosomiasis in humans: Schistosoma (S.) mansoni, S. japonicum and S. haematobium. S. mansoni, endemic to Africa, the Caribbean and South America, and S. japonicum, the Asian schistosome, cause intestinal schistosomiasis. S. haematobium inha- bits Africa and the eastern Mediterranean and causes ARTICLE IN PRESS www.elsevier.de/imbio 0171-2985/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2008.11.017 Corresponding author at: Department of Zoology, Faculty of Science, Beni-Sueif University, Beni-Sueif, Egypt. Tel.: +20 12 3749077; fax: +20 82 2334551. E-mail address: [email protected].
Transcript
ARTICLE IN PRESS
Immunobiology 214 (2009) 712–727
0171-2985/$ - se
doi:10.1016/j.im
�CorrespondiScience, Beni-Su
fax: +2082233
E-mail addr
www.elsevier.de/imbio
Immunomodulatory effects of curcumin treatment on murine
schistosomiasis mansoni
Gamal Allama,b,�
aDepartment of Zoology, Faculty of Science, Beni-Sueif University, Beni-Sueif, EgyptbCollege of Medicine and Medical Sciences (CMMS), Taif University, Taif, Saudi Arabia
Received 7 October 2008; received in revised form 28 November 2008; accepted 29 November 2008
Abstract
Curcumin is a polyphenol derived from the dietary spice turmeric. It has been shown to regulate numeroustranscription factors, cytokines, adhesion molecules, and enzymes that have been linked to inflammation. In additionto inhibiting the growth of a variety of pathogens, curcumin has been shown to have nematocidal activity. The presentstudy was designed to evaluate the schistosomicidal activity of curcumin in vivo as well as immunomodulation ofgranulomatous inflammation and liver pathology in acute schistosomiasis mansoni. Mice were infected each with80 Schistosoma (S.) mansoni cercariae and injected intraperitoneally with curcumin at a total dose of 400mg/kg bodyweight. Curcumin was effective in reducing worm and tissue-egg burdens, hepatic granuloma volume and liver collagencontent by 44.4%, 30.9%, 79%, and 38.6%, respectively. Curcumin treatment restored hepatic enzymes activities tothe normal levels and enhanced catalase activity in the liver tissue of infected mice. Moreover, hepato-spleenomegalyand eosinophilia induced by S. mansoni infection were largely improved with curcumin treatment. Infected micetreated with curcumin showed low serum level of both interleukin (IL)-12 and tumor necrosis factor alpha (TNF-a),but IL-10 level was not significantly altered. Specific IgG and IgG1 responses against both soluble worm antigen(SWAP) and soluble egg antigen (SEA) were augmented with curcumin treatment, but IgM and IgG2a responseswere not significantly changed. In conclusion, curcumin treatment modulates cellular and humoral immune responsesof infected mice and lead to a significant reduction of parasite burden and liver pathology in acute murineschistosomiasis mansoni.r 2008 Elsevier GmbH. All rights reserved.
are at risk for schistosomiasis, with 207 million infectedin 76 countries and territories (Steinmann et al., 2006;Lammie et al., 2006). There are three main schistosomespecies causing schistosomiasis in humans: Schistosoma
(S.) mansoni, S. japonicum and S. haematobium.
S. mansoni, endemic to Africa, the Caribbean and SouthAmerica, and S. japonicum, the Asian schistosome,cause intestinal schistosomiasis. S. haematobium inha-bits Africa and the eastern Mediterranean and causes
ARTICLE IN PRESSG. Allam / Immunobiology 214 (2009) 712–727 713
urinary schistosomiasis (Hu et al., 2004). Variousmethods have been used to combat schistosomiasis,including improving sanitary conditions, but to littleavail, in part owing to the lack of effective vaccinesagainst the parasites (Gryseels, 2000). Currently, che-motherapy is the control strategy used. Only one drug,praziquantel, is currently effective against all species ofschistosome, and it has a key role in population-baseddisease-control programs in most endemic countries(Salvioli et al., 1997; WHO, 1997). However, praziquan-tel does not prevent re-infection, is inactive againstjuvenile schistosomes and only has a limited effect onalready developed liver and spleen lesions, as well as onthe emergence of schistosome phenotypes that areresistant to praziquantel chemotherapy (Fallon andDoenhoff, 1994; Stelma et al., 1995; Ismail et al.,1999). Furthermore, the use of such drug for treatmentis complicated due to the difficulties and expensesinvolved in a long-lasting maintenance of these pro-grams (Boros, 1989). Metrifonate and oxamniquine areless widely used due to a lack of easy availability, poorcure rates in the field and the demonstration of drug-induced resistance in laboratory models (Cioli et al.,1993; Brindley, 1994; Cioli, 1998). It is therefore clearthat in order to ensure the effective drug control ofschistosomiasis in the future, the identification of newand effective schistosomicidal compounds is essential(Sanderson et al., 2002).
Curcumin is a naturally occurring phenolic com-pound isolated as a yellow pigment from turmeric (dryrhizomes of curcumin longa) which is commonly used asa spice and food colorant (Buescher and Yang, 2000).This compound has been reported to possess a variety ofbiological and pharmacological activities, includingantioxidant (Yadav et al., 2005; Wei et al., 2006), anti-inflammatory (Chainani-Wu, 2003; Woo et al., 2007),anticarcinogenic (Mukhopadhyay et al., 2002; Aggarwalet al., 2003; Devasena et al., 2003, 2007), antidiabetic(Srinivasan and Menon, 2003), anti-HIV (Jordan andDrew, 1996; Barthelemy et al., 1998), anti-bacterial(Chopra et al., 1941; Bhavani Sankar and Murthy,1979), anti-Leishmanial (Araujo et al., 1998, 1999;Koide et al., 2002), anti-Trypanosomal (Nose et al.,1998), and anti-malarial activities (Rasmussen et al.,2000; Reddy et al., 2005). In addition to inhibiting thegrowth of a variety of pathogens, curcumin has beenshown to have nematocidal activity against Toxocara
canis (Kiuchi et al., 1993; Araujo and Leon, 2001).The pathology associated with schistosomiasis man-
soni is largely attributed to the intense granulomatousinflammations and subsequent fibrosis induced byparasite eggs that become trapped in host organs suchas the liver and intestine. Granuloma formation isdependent on CD4+ T cell responses (Warren et al.,1967; Iacomini et al., 1995; Hernandez et al., 1997). Inmurine models of schistosomiasis, schistosome eggs and
egg-derived antigens are potent and independent in-ducers of type 2 T cell responses (Grzych et al., 1991;Vella and Pearce, 1992). Induction of type 2 responsesby parasite eggs after the onset of oviposition thusaccounts for a skewing of systemic T cell responsesduring schistosome infection, from a type 1 responseduring prepatency which is characterized by theproduction of IFN-g to a type 2 response by 8 weekspost-infection which is characterized by the secretion ofIL-4, IL-5, IL-13 and IL-10 (Grzych et al., 1991; Pearceet al., 1991; Sher et al., 1991; Hernandez et al., 1997).Granuloma formation therefore occurs in an environ-ment that is initially proinflammatory and type 1-like,but which subsequently polarizes rapidly to one that ispredominantly type 2-like (Davies et al., 2004). Therelative influence of Th1 and Th2 cytokines on thepathogenesis of schistosomiasis remains controversial inboth mice and humans. Nevertheless, decreased fibrosisin the murine model is associated with diminishedproduction of type 2 cytokines and increased productionof type 1 cytokines (Cheever and Yap, 1997). However,in the absence of IL-4 (key cytokine driving the Th2response), some strains of mice overproduce several pro-inflammatory mediators, including IFN-g, TNF-a andnitric oxide (NO), leading to an acutely fatal diseasefollowing infection (Brunet et al., 1997). It has beenreported that up to 30% mortality is observed in IL-10-deficient mice at the acute stage post-infection(Hoffmann et al., 2000). Moreover, in IL-4-deficientanimals, liver damage caused by excess production ofTNF-a, NO and reactive oxygen intermediates appearsto be the primary cause of mortality (La Flamme et al.,2001). Therefore, modulation of both Th1 and Th2responses could be downregulate granulomatous in-flammation and consequently reduce pathology ofschistosomiasis.
Curcumin has been shown to be a potent immuno-modulatory agent that can modulate the activation ofT cells (Cipriani et al., 2001; Ranjan et al., 1998a, 2005),B cells (Ranjan et al., 1998b; Churchill et al., 2000),macrophages (Bhaumik et al., 2000; Li and Liu, 2005),neutrophils (Jagetia and Aggarwal, 2007), natural killercells (Gao et al., 2004; Yadav et al., 2005), and dendriticcells (Kim et al., 2005). Curcumin can also downregulatethe expression of various proinflammatory cytokinesincluding IL-1(Chan, 1995; Terry et al., 1998; Abe et al.,1999; Lee et al., 2003), IL-2 (Ranjan et al., 2004; Yadavet al., 2005; Siddiqui et al., 2006), IL-6 (Renard et al.,2001; Kim et al., 2005; Gulcubuk et al., 2006), IL-8(Renard et al., 2001; Wang et al., 2006), IFN-g (Kanget al., 1999b; Gao et al., 2004), monocyte inflammatoryprotein-1a and monocyte chemotactic protein-1 (Abeet al., 1999) as well as inhibition of inducible nitric oxidesynthase (iNOS) expression (Chan et al., 1998), and NOproduction (Brouet and Ohshima, 1995; Yadav et al.,2005). Moreover, Curcumin inhibits the proliferation of
ARTICLE IN PRESSG. Allam / Immunobiology 214 (2009) 712–727714
lymphocytes and their ability to secrete IL-4, IL-5, andGM-CSF (Kobayashi et al., 1997).
Taken together, these findings warrant further con-sideration of curcumin as an immuomodulator forimmune disorders induced by schistosomiasis. Theprimary aim of the present work was to investigate theimmunoregulatory role of curcumin on granulomaformation. A secondary aim was to determine the effectof curcumin administration on the survival and fecund-ity of adult S. mansoni as well as liver pathologyresulting from such infection.
Materials and methods
Mice and parasite
Male CD1 albino mice were purchased from theSchistosome Biological Supply Center, TheodoreBilharz Research Institute (TBRI, Imbaba, Giza,Egypt). Mice were maintained under suitable livingconditions. S. mansoni (John Bruce Egyptian strain)cercariae were obtained from infected Biomphalaria
alexandrina snails (TBRI) as previously described bySchubert (1948).
Experimental design
Curcumin (Riedel De Haen Ag., Seelze-Hannover,Germany) was dissolved in 4.5% dimethyl sulfoxide(DMSO; Sigma Chemical, St. Louis, MO). Both naive(NT) and infected (IT) mice were injected intraperito-neally with curcumin as absorption seems to be higherby this route than after oral administration (Pan et al.,1999; Dohare et al., 2008; Moon et al., 2008), at a totaldose of 400mg/kg body weight divided equally into16 injections (two injections/week for 8 consecutiveweeks, starting from the first week of the infection). Thedose of curcumin (400mg/kg body weight) was selectedon the basis of our pilot study and several publisheddata reviewed in Aggarwal and Harikumar (2009).Control groups consisted of a normal untreated (NU)and an infected untreated (IU) group, were given thesame amount of the vehicle at the same time as treatedgroups.
Infection and worm recovery
Mice were infected with S. mansoni live cercariaeaccording to Stirewalt and Bronson (1955) and Bruceand Radke (1971) with some modifications. Briefly,mice were individually placed in mouse retrainingchambers, allowing the tail outside the chamber. Aftercleaning the tail with dechlorinated tap water, it wasinserted into a vial containing 80 cercariae in 2ml
dechlorinated tap water and located into an exposureboard. Mice were left in contact with the infectivecercariae for 1 h (hour), and then removed from thechamber and tails were allowed to dry. Remainingcercariae were counted and mice receiving less than 95%of the cercariae were excluded from the experiment.At the end of the 8th week post-infection all mice weresacrificed and worm burdens were estimated by portalperfusion according to Duvall and DeWitt (1967).
Blood and tissue sampling
Before perfusion, blood was collected by orbitalpuncture in heparinised tubes and dry clean tubes. Serawere collected from the clotted blood samples aftercentrifugation at 400� g for 15min (min) at 4 1C, thendivided into aliquots and stored at �70 1C until used.The liver from each mouse was excised immediatelyafter perfusion and rinsed with ice-cold saline solution.The ventral median lobe of the liver was fixed in 10%neutral buffered formalin for histopathological study.Another portion was homogenized in 0.9% saline,centrifuged at 400� g for 15min and the supernatantwas decanted and stored at �70 1C until use for differentbiochemical estimations.
Total and differential leukocyte count
Total white blood cells (WBCs) count was carried outin heparinised-blood samples by using Turk’s solutionand Neubauer haemocytometer. Differential leukocytecount was performed in Giemsa-stained blood smearsaccording to Dacie and Lewis (1991).
Egg burden estimation
The number of S. mansoni eggs in the perfused liverand in the intestine of mice was estimated after alkalidigestion as described by Cheever (1968). The mean eggsper gram (g) tissue (liver and intestine) and mean eggs/gtissue per possible worm pairs were calculated using thefollowing formula:
Liver tissues were fixed in buffered neutral formalinand processed to make paraffin blocks. The blocks werecut into 4 mm sections, sections stained with hematoxylinand eosin (H&E) were used for the determination of
ARTICLE IN PRESSG. Allam / Immunobiology 214 (2009) 712–727 715
granuloma size (Ohmae et al., 1991) and sections stainedwith Masson’s triple (Masson’s trichrome) stain wereused for collagen fibres (Bancroft and Stevens, 1982).The diameters of granulomas surrounding a singlemature schistosome egg were measured in liver sectionsby using an ocular micrometer (Carl Zeiss, Thornwood,NY, USA). Twenty granulomas per mouse weremeasured and the largest diameter and that perpendi-cular to it were averaged. The volume (mm3) of eachgranuloma was calculated assuming a spherical shapeusing the following formula: Volume of sphere ¼ 3pR3
(the radius ‘‘R’’ was obtained by dividing the maindiameter of the lesion by two) and the mean volume foreach group was calculated (Mahmoud and Warren,1974).
Hepatic collagen content determination
The amount of collagen in liver samples wasdetermined from its hydroxyproline content by themethod of Neuman and Logan (1950a, b). Briefly, thedefatted and dried samples were hydrolyzed by auto-claving in 2ml of 6N HCl at 50 pounds pressure. Theprotein hydrolysates were used for the determination ofhydroxyproline and the percentage of collagen of tissuesample was calculated by using the 7.46 factor ofNeuman and Logan (1950a) and Baykal-Erkilic et al.(1995). Meanwhile, a standard curve was constructed inorder to determine the reproducibility of the assay.
Estimation of hepatic enzymes activities
Hepatic alanine aminotransferase (ALT) and aspar-tate aminotransferase (AST) activities in the homoge-nate supernatant were estimated according to the kineticmethod of Bergmeyer et al. (1978), using reagent kitspurchased from Spinreact Company (Spain). Hepaticg-glutamyl transferase (g-GT) was determined accordingto the kinetic method of Tietz (1991) and Young (1995)using reagent kits obtained from BioSystems (Spain).Liver lactate dehydrogenase (LDH) activity was deter-mined according to the method described by Tietz(1986), using reagent kits purchased from Dia Lab(Vienna, Austria). Liver catalase was determined ac-cording to the technique of Cohen et al. (1970) usingreagents prepared in the laboratory. Results of catalaseactivity were expressed in terms of the first-orderreaction rate constant (k) as indicated by Cohen et al.(1970).
Cytokine profile analysis
Sandwich enzyme-linked immunosorbent assay(ELISA) was used to measure serum cytokines levels.Cytokine concentrations for mouse IL-4, IL-10, IL-12
+p40 and TNF-a were determined with commerciallyavailable reagents and ELISA kits purchased fromBioSource International (Camarillo, CA, USA). Cyto-kines concentrations were determined using a standardcurve obtained from the known concentration ofcytokine standards included in each assay plate accord-ing to manufacturer instructions.
Detection of S. mansoni-antigens specific antibodies
S. mansoni antigens preparations, soluble wormantigen (SWAP) and soluble egg antigen (SEA), werepurchased from TBRI. Antigens were suspended in0.01M phosphate buffered saline (PBS), pH 7.2 andprotein content was measured by Lowry’s method(Lowry et al., 1951). The measurement of specific anti-SWAP or anti-SEA antibodies was performed usingindirect ELISA. Maxisorp 96-well microtiters plates(Nunc, Denmark) were coated with 5 mg/ml of eitherSWAP or SEA in 0.05M carbonate–bicarbonate buffer,pH 9.6, for 12–16 h at 4 1C, then blocked for 1 h at 37 1Cwith 200 ml per well of PBS, pH 7.2, with 0.05% Tween20 (PBST) plus 10% fetal bovine sera (FBS). A 100ml ofeach serum diluted 1:100 in PBST was added per welland incubated for 2 h at 37 1C in a humidified atmo-sphere. Biotionylated rat anti-mouse IgM, total IgG,IgG1, and IgG2a (pharMingen, San Diego, CA) wereused for the detection of IgM, IgG, IgG1, and IgG2a,respectively, at a dilution of 1:500 in PBST andincubated for 1 h at 37 1C in a humidified atmosphere.Unbound biotine was removed by 5 times washingwith PBST. Then peroxidase-labeled streptavidin(pharMingen) diluted 1:4000 in PBST and added100 ml/well for 1 h at room temperature. Unboundconjugate was removed by 5 times washing with PBST,and the extant of binding was monitored colorometri-cally using equal volumes of 3, 30, 5, 50-tetramethylbenzidine (TMB) and H2O2 (Kirkegaard and PerryLaboratories Inc., Gaithersburg, MD) as a peroxidasesubstrate. The reaction was allowed to proceed for10min, after which absorbance was read at wavelengthof 650 nm using a microplate reader (Bio-Rad, Hercules,CA). Individual mouse sera were applied triplicate, andthe mean was calculated. Data were presented as meanoptical density (O.D.).
Statistical analysis
The data were analyzed using one way analysis ofvariance (ANOVA) followed by LSD analysis tocompare various groups with each other (PC-STAT,University of Georgia, 1985). Results were expressed asmean7standard deviation (SD) and values of p40.05were considered statistically insignificant, while thoseof po0.05 and po0.01 were considered statistically
ARTICLE IN PRESSG. Allam / Immunobiology 214 (2009) 712–727716
significant and highly significant, respectively. F-prob-ability for each variable expressed the general effectbetween groups.
Results
Body weight and relative organ weight percentage
In order to investigate the effect of curcumintreatment on the body weight gain of both normal andS. mansoni-infected mice, body weight was measuredbefore and after 8 weeks of treatment. Before experi-ment, the range of mean body weight of mice in thedifferent groups was 20–2171.0–1.2 g and was notsignificantly (p40.05) different between tested groups.At 8 weeks post-infection and/or post-treatment,curcumin administration significantly (p40.01) in-creased the body weight of infected mice and had nosignificant (p40.05) effect on body weight of non-infected mice as compared to the control ones (Table 1).
To examine the effect of curcumin administration onhepatomegaly and spleenomegaly induced by S. mansoni
infection, both liver and spleen were excised fromdissected mice after perfusion, weighed and relativeweight percentage in relation to body weight werecalculated. As shown in Table 1, there was no significant(p40.05) difference in relative liver and spleen weightsof both NU and non-infected treated (NT) mice groups.However, IU mice showed a highly significant (po0.01)increase in both relative liver and spleen weights ascompared with NU mice. On the other hand, infectedtreated (IT) mice showed a highly significant (po0.01)
Table 1. Effect of curcumin treatment on body weight, relative or
Groups Parameters
Body
weight
(g)
Relative
liver
weight
Relative
spleen
weight
WBCs
count/mm
NU 30.8872.1a 4.2470.28c 0.4570.08c 44657517
NT 29.6271.1a 4.4270.90c 0.3870.08c 45007430
IU 26.2371.8b 9.0870.65a 1.4070.28a 39057395
IT 29.0171.2a 6.8071.23b 0.6970.13b 43707540
F-value 24.67 41.40 39.80 1.68
F-Probabil. po0.01 po0.01 po0.01 p40.05
LSD at 5% 2.12 1.13 0.22 –
LSD at 1% 2.94 1.56 0.31 –
At the end of the 8th week post-infection and/or post-treatment non-infected u
infected treated (IT) mice were weighted before sacrifice. Liver and spleen w
Relative liver and spleen weights were calculated as the following:
Relative organ weight ¼ (absolute weight/body weight)� 100.
Blood was collected at the time of sacrifice and diluted 1:20 in Turk’s solutio
Neubauer heamocytometer. Percentage of different types of leukocytes wa
microscope. Data are expressed as mean7SD of 10 mice per each group. V
decrease in both relative liver and spleen weights ascompared to the IU group. This result indicates thatcurcumin treatment alleviate hepatosplenomegaly in-duced by S. mansoni infection.
Total and differential leukocyte count
To explore the effect of curcumin treatment on theperipheral-blood leukocytes, blood samples were ob-tained immediately before sacrifice in heparinised tubes.Blood was diluted 1:20 with Turk’s solution and WBCswere enumerated by using Neubauer chamber. Asdepicted in Table 1, there was no significant (p40.05)difference in total leukocyte count between the testedmice groups.
Different types of leukocytes were counted in thinblood smears stained with Giemsa. As shown in Table 1,there was no significant (p40.05) effect of curcumintreatment on neutrophil, lymphocyte, monocyte, oreosinophil count of the NT mice, compared to the NUmice. Similarly, the IT mice showed no significant(p40.05) difference in both neutrophil and monocytecount, compared to the IU mice. Lymphocyte count ofIT mice significantly (po0.01) increased, but eosinophilcount highly significantly (po0.01) decreased, comparedto the IU mice. On the other hand, percentage of bothneutrophil and eosinophil of IU mice highly (po0.01)significantly increased, while lymphocyte percentagesignificantly (po0.01) decreased, compared to the NUmice. Monocyte count was not significantly (p40.05)different from the IU and NU mice groups. Basophilswere not observed in all examined blood smears.
gans weight, total and differential leukocyte count.
3Differential leukocyte count
Neutrophil
%
Lymphocyte
%
Monocyte
%
Eosinophil
%
a 1473.1b 7673.1a 671.58a 2.670.89b
a 20.476.7b 73.275.0a 4.272.68a 371.7b
a 46.474.9a 4374.1c 3.471.51a 8.873.9a
a 42.673.4a 5173.3b 373.3a 3.471.5b
47.12 65.02 1.53 7.8
po0.01 po0.01 p40.05 po0.01
6.37 5.32 – 3.13
8.78 7.33 – 4.31
ntreated (NU), non-infected treated (NT), infected untreated (IU), and
ere excised from perfused mice and absolute weights were estimated.
n. Leukocytes were count under 40� light microscope using improved
s estimated in Giemsa stain blood smear using oil immersion light
alues not sharing common superscripts denote significant differences.
ARTICLE IN PRESS
Table 2. Effect of curcumin administration on worm and egg burdens, granuloma volume and liver collagen content.
Groups Parameters
Worm burden Egg burden Granuloma
volume
(mm3� 103)
Collagen
(mg/100mg)Male Female Total PWP Eggs/g
tissues
Eggs/g
tissues/PWP
IU 14.573.9a 15.773.7a 30.277.5a 14.573.9a 31329.574421a 294371507a 25.5673.58a 6.5370.64a
IT 8.272.7b 8.774.7b 16.876.8b 7.273.1b 2165976852b 277971414a 5.3670.53b 4.0170.86b
Both infected untreated (IU) and infected treated (IT) mice were infected each with 80 cercariae and perfused 8 weeks post-infection. After perfusion
has been completed, worm were collected and counted and possible worm pairs (PWP) were calculated. S. mansoni eggs were estimated in both liver
and intestine tissues after digestion in 5% KOH and eggs/g tissues and eggs/g tissues per PWP were calculated. Hepatic granuloma volume was
measured in H & E stained liver sections and liver collagen content was estimated in liver homogenate. Percentage of reduction in all parameters was
calculated according the following equation:
% reduction ¼ (value of IU�value of IT/value of IU)� 100.
Data are expressed as mean7SD of 10 mice per each group. Values not sharing common superscripts denote significant differences.
G. Allam / Immunobiology 214 (2009) 712–727 717
Worm burden
In an attempt to determine the schistosomicidal effectof curcumin in vivo, CD1 mice infected each with 80S. mansoni cercariae and treated with a total dose of400mg/kg body weight, divided into 16 injections,2 times per week, started from the 1st week post-infection to the 8th week post-infection. Mice perfusedat the end of the 8th week post-infection and wormburden was estimated. As shown in Table 2, curcumintreatment significantly reduced male (po0.01), female(po0.05), total worms (po0.01), and possible wormpairs (po0.01) counts by 43.5%, 44.5%, 44.4%, and50.3%, respectively, compared to the IU mice.
Egg burden
To evaluate the potential effect of curcumin on wormfecundity and tissue egg loads, both liver and intestinetissues were digested separately in 5% KOH and eggs/gtissues as well as eggs/g tissue per possible worm pairswere calculated. As depicted in Table 2, eggs/g tissues wassignificantly (po0.05) decreased by 30.9% with curcumintreatment, compared to the IU group. However, eggs/gtissue per possible worm pairs not significantly (p40.05)changed between IT and IU mice. This result means thatcurcumin treatment not alter worm fecundity.
Granuloma volume and liver histopathology
To investigate the effect of curcumin on granulomatousinflammation, hepatic granuloma volume was measuredin H&E stained liver section at 8 weeks post-infections.
Curcumin administration highly significantly (po0.01)reduced granuloma volume in IT mice by 79%, comparedto the IU mice (Table 2). Histological examination ofH&E stained liver sections showed that granulomas of IUmice was composed of central ova surrounded bylaminated layers of fibrous tissue associated with inflam-matory cells at the periphery. Also, severe necrosis wasseen in the hepatic tissue of the IU mice (Fig. 1A).However, granuloma of the IT mice was seen as aconcentric focus of mononuclear and polymorphonuclearcells around the egg and the laminated layers of fibrousconnective tissue nearly disappeared. Minimal microvas-cular changes and no hepatocyte necrosis were noticed inthe liver sections of IT mice (Fig. 1B).
Hepatic collagen
In order to examine the anti-fibrotic effect ofcurcumin, collagen in liver tissue was quantified fromits hydroxyproline content. After defatting and drying,liver tissues were hydrolyzed by autoclaving in 6N HClat 50 pounds pressure. The hydrolysates were used forthe determination of hydroxyproline and the percentageof collagen. As shown in Table 2, hepatic collagencontent significantly (po0.01) decreased in the IT miceby 38.6%, compared to the IU mice.
Liver sections stained with Masson’s triple stain todemonstrate the effect of curcumin on collagen fibresdeposition. Examination of liver sections of the IU miceshowed hepatic granulomas with centrally placed ovasurrounded by dense bundles of collagen fibres, whichappear in green color, with interstitial and perivascularcollagen deposition (Fig. 1C). On the other hand, in theIT mice, the periovular collagen fibres markedly
ARTICLE IN PRESS
Fig. 1. Photomicrographs of hepatic granulomas of 8 weeks infected untreated (A, C) and infected treated mice with curcumin (B,
D). Liver sections stained with hematoxylin and eosin (A, B) showed granuloma volume and liver necrosis. Sections stained with
Masson’s triple stain showed collagen fibres, green in color (C, D).
G. Allam / Immunobiology 214 (2009) 712–727718
decreased in amount with thinner loosely arrangedfibres. Most of granulomas in IT mice contained veryfine collagen fibres with a minimal interstitial andperivascular collagen deposition (Fig. 1D).
Hepatic enzymes activities
To evaluate the ameliorative effect of curcumintreatment on liver pathology induced by S. mansoni
infection, activity of ALT, AST, g-GT, and LDH weremeasured in liver tissue homogenate supernatant at8 weeks post-infection. As shown in Table 3, there wasno significant difference (p40.05) in the activity of allhepatic enzymes between the NT and NU mice. Allenzymes activities of the IU mice were significantly(po0.01) lower than those of the NU and NT groups.However, curcumin treatment restored the liver enzymesactivities of infected mice to the normal level. This
ARTICLE IN PRESS
Table 3. Effect of curcumin administration on the activities of some enzymes in liver tissue homogenate.
NU 403.6758.9a 246.9719.8ab 65.9712.1a 43.478.2ab 45.3710.8b
NT 352.6767.1ab 271.6755.1a 55.979.2a 47.279.1a 45.777.5b
IU 243.4716.8c 143.2745.0c 31.5710.9b 30.473.7c 43.979.2b
IT 313.9763.6b 200730.4b 50.1710.5a 37.575.7bc 66.575.6a
F-value 8.89 14.69 8.56 5.43 7.2
F-Probabil. po0.01 po0.01 po0.01 po0.01 po0.01
LSD at 5% 66.79 46.79 12.92 9.39 15.4
LSD at 1% 91.14 63.80 17.71 12.87 21.2
Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), g-glutamyl transferase (g-GT), lactate dehydrogenase (LDH), and
catalase were measured in liver tissue homogenate supernatant of non-infected untreated (NU), non-infected treated (NT), infected untreated (IU),
and infected treated (IT) mice at the 8th week post-infection and/or post-treatment. Data are expressed as mean7SD of 10 mice per each group.
Values not sharing common superscripts denote significant differences.
0
50
100
150
200
250
300
IL-10
pg/m
l
NUNTIUIT
b b
a
a
bb
a
b
c c
a
b
TNF-alphaIL-12
Fig. 2. Effect of curcumin treatment on serum levels of IL-10,
IL-12 and TNF-a. Blood was collected at the 8th week post-
infection and/or post-treatment from non-infected untreated
(NU), non-infected treated (NT), infected untreated (IU), and
infected treated (IT) mice. Cytokines levels were measured by
sandwich ELISA. Data represented as the mean7SD of 10
mice. Columns not sharing common superscripts denote
significant differences. po0.05 (IL-12), po0.01 (IL-10 and
TNF-a).
G. Allam / Immunobiology 214 (2009) 712–727 719
indicated by the highly significant (po0.01) increase inthe ALT, AST, and g-GT activities of the IT mice incomparison with the IU mice. There was no significantdifference in the activities of AST, g-GT, and LDHbetween the IT and NU mice.
To explore the effect of curcumin on antioxidantenzymes, catalase activity was measured in liver tissuehomogenate. As shown in Table 3, the NT mice showedno significant difference (p40.05) in the activity ofcatalase, compared to the NU mice. However, the ITmice revealed a high significant (po0.01) increase incatalase activity, compared to the IU mice.
Cytokine profile
To investigate the immunomodulating effects ofcurcumin, IL-10, IL-12 and TNF-a were quantified inserum at 8 weeks post-infection and/or post-treatmentusing sandwich ELISA. As depicted in Fig. 2, level ofIL-10 not significantly changed (p40.05) in the NTmice, compared to the NU mice. Also, IL-10 level of theIT mice was not significantly (p40.05) different fromthat of the NU mice. However, the IU and IT miceshowed a significant (po0.01) increase in IL-10 level,compared to the NU and NT mice.
On the other hand, IL-12 level of IT mice notsignificantly differed (p40.05) from those of the NU orNT mice, but significantly (po0.05) decreased, com-pared to that of the IU mice. S. mansoni-infected miceshowed a high significant (po0.05) level of IL-12 incomparison with the other groups (Fig. 2).
The pattern of TNF-a serum level was similar to thatof IL-12. There was no significance (p40.05) differencein TNF-a level of the NT and NU mice. However, ITmice showed a high significant (po0.01) decrease inTNF-a level, compared to the IU mice. TNF-a serumlevel of IU mice was the highest one in comparing withthe other groups (Fig. 2).
Unfortunately, IL-4 was undetectable in the sera of alltested mice groups despite of we used 100 ml of serumwithout dilution for cytokine detection.
Specific antibody response
Since curcumin treated animals demonstrated a high-er reduction in parasite burden, we decided to determinethe specific humoral immune response induced ininfected animals following treatment. Therefore, wemeasured the total IgG, IgG1, IgG2a, and IgM in
ARTICLE IN PRESS
00.20.40.60.8
11.21.41.61.8
O.D
.
IUIT**
**
*
**
SWAP SEAIgG IgG1 IgG2a IgM IgG IgG1 IgG2a IgM
Fig. 3. Effect of curcumin treatment on antibody response of
S. mansoni-infected untreated (IU) and infected treated (IT)
mice against soluble worm antigen (SWAP) and soluble egg
antigen (SEA) preparations at 8 weeks post infections. Serum
was collected at the time of sacrifice and S. mansoni antigens-
specific total IgG, IgG1, IgG2a, and IgM levels were
determined by indirect ELISA. Data reported as the mean
optical density (O.D.)7SD of 10 mice. *po0.05, **po0.01 vs.
corresponding IU value.
G. Allam / Immunobiology 214 (2009) 712–727720
response to SWAP and SEA at 8 weeks post-infectionusing indirect ELISA. The data depicted in Fig. 3showed that both total IgG and IgG1 of the IT micesignificantly increased in response to both SWAP andSEA, compared to the IU mice. However, response ofIgG2a and IgM against either SWAP or SEA notsignificantly (p40.05) altered between tested groups.IgG2a level in response to both SWAP and SEA was thelowest response among studied isotypes and sub-isotypes. Curcumin augmented response of IgG1 againsteither SWAP or SEA. Therefore, the IgG1/IgG2a ratiowas increased with curcumin treatment.
Discussion
Many plant species have been used throughout theworld in traditional medicine for the treatment ofboth veterinary and human helminths (Hammondet al., 1997; Fennell et al., 2004) but few plants havebeen screened for activity against adult S. mansoni
(Sanderson et al., 2002). The present study investigatedthe schistosomicidal activity of curcumin in vivo. Thedata presented here showed that curcumin treatmentreduces both male and female worm burdens by 43.5%and 44.6%, respectively. It appears that this compoundis active against both male and female worms, not likeother drugs which act preferentially against one genderrather than the other. The possible explanations for thisfinding are: first, curcumin may have a direct toxiceffective against adult S. mansoni such as that wasreported against Toxocara canis (Kiuchi et al., 1993;Araujo and Leon, 2001). But, the mechanism by which
curcumin might function as a schistosomicidal agent isunclear and needs further investigation. Second, curcu-min may be induced an effective immune responseagainst S. mansoni infection. The data of the presentstudy showed that curcumin treatment augmented bothIgG and IgG1 responses against both SWAP and SEA.It has been found that curcumin enhances the humoralimmune response by increasing IgG production(Kuramoto et al., 1996). The absolute requirement forantibody response in protection against murine andhuman schistosomiasis has been amply documented(Hoffmann et al., 1999; Jankovic et al., 1999; Dunne andMountford, 2001). The effective factors in protectivehumoral immune response appear to be depended onIgG antibodies (Mangold and Dean, 1986), particularlythose of the IgG1 isotype. Passive transfer of differentisotypes fractionated from whole protective serumrevealed that, only IgG1 has the capacity to protectnaive recipients against challenge infection. The resis-tance transferred by IgG1 represented more than 60%of that obtained with whole serum (Delgado andMcLaren, 1990; King et al., 1996). Moreover, micevaccinated with recombinant P44 (fructose 1,6 bispho-sphate aldolase antigen) were able to develop high levelsof IgG and IgG1 and displayed low levels of IgG2aisotype with a significant protection of 57% against achallenge infection and significant decrease in hepaticgranuloma formation (Marques et al., 2008). Theantibody may impair larval viability and migration byinteraction with critical sites in surface membrane-exposed molecules of importance for the parasite suchas the glucose transporter, the amino acid permease andenzymes necessary for the generation of energyand membrane repair. Such ‘‘neutralizing’’ antibodiesmight even interfere with the welfare of adultworms in the liver (El Ridi, 2002). It has been shownthat in rat, primate and human schistosomiasis,antibody-dependent cell-mediated cytotoxicity (ADCC)is the main mechanism of killing parasite larvae (Capronand Capron, 1994). In mice, cytotoxicity in schistoso-miasis requires also activated macrophages (James et al.,1983, 1984). Curcumin has been found to differentiallyactivate macrophages and increases its phagocyticactivities (Bhaumik et al., 2000; Li and Liu, 2005).
Enormous eggs laid by the mature worm stay in theliver and other tissues and lead to severe syndromeincluding inflammation, granuloma, and tissue fibrosis.Therefore, reducing eggs load in the tissues cansignificantly relieve the syndrome of schistosomiasis(Huang et al., 2005). The present study demonstratedthat curcumin administration decreases tissue egg loadby 39.9%. However, eggs/g tissue per worm pairswas decreased by only 5.6% and this reduction wasnot significantly different from IU mice. This meansthat curcumin not affect adult worm fecundity andthe decrease in tissue eggs load could be attributed to
ARTICLE IN PRESSG. Allam / Immunobiology 214 (2009) 712–727 721
the decrease in total worm burden and possible wormpairs.
Cytokines are important regulators of immunoin-flammatory responses and play a major role in theregulation of collagen fibres deposition and degradation(Vaillant et al., 2001). The data of the presentinvestigation showed a decrease in serum level bothTNF-a and IL-12 of infected treated mice. This findingrunning parallel with the previous reports on otherexperimental models which stated that, curcumin down-regulate the expression and production of both TNF-a(Gaddipati et al., 2003; Lantz et al., 2005; Siddiqui et al.,2006; Lee et al., 2003, 2008) and IL-12 (Kang et al.,1999a, b; Kim et al., 2005) through inhibition of thetranscription factor nuclear factor kappa B (NF-kB)responsive genes involved in the production of thesecytokines (Gao et al., 2004). However, IL-10 notsignificantly affect by curcumin treatment either ininfected or non-infected animals. There is a contra-diction in the previous studies concerning the effect ofcurcumin treatment on IL-10 expression. Gaddipatiet al. (2003) reported that curcumin treatment sup-presses the production of IL-10 mRNA at 2 and 24 hafter hemorrhage/resuscitation and this action ismediated through the inhibition of NF-kB activationand activation of protein-1 (AP-1). On the other hand,other studies reported that curcumin enhances theexpression of IL-10 mRNA in different cell lines(Grandjean-Laquerriere et al., 2002; Sharma et al.,2007). Our result comes in accord with the previousstudy of Zhang et al. (2006) whom stated that curcumintreatment of chronic colitis led to an increase in theexpression of IL-10 mRNA in colon mucosa but couldnot increases the level of IL-10 in serum. The presentstudy showed that curcumin treatment increases theIgG1/IgG2a ratio. IgG1 is known to be a Th2-dependentimmunoglobulin isotype whereas IgG2a is Th1-dependentisotype (Finkelman et al., 1990). Consequently, IL-10,IL-12, TNF-a, and the IgG1/IgG2a profiles observed inthis study following administration of curcumin isindicative of a dominant Th2-type immune response.However, the exact effect of curcumin on Th1/Th2 biasduring schistosomiasis remains obscured and needsfurther investigation.
Due to its low toxicity, curcumin is currently underconsideration as a potential therapeutic agent to variouschronic illnesses in which inflammation is known to playa major role (Aggarwal and Harikumar, 2009).A granulomatous reaction around eggs within the liverand other organs is the pathology characteristic ofschistosomiasis. This inflammatory process leads to thedestruction of an extensive area of the organ where itoccurs (Bergquist, 2002). Besides reduction in worm andegg burdens, if granuloma formation could be preventedor suppressed, the development of severe disease mightbe averted. The data of the present study showed that
curcumin treatment reduces granuloma volume by 79%.Previous studies have indicated that disease develop-ment is affected by the interleukins, which regulate thegranulomatous response especially IL-10 (Wynn et al.,1998), IL-12 (Wynn et al., 1995), and TNF-a (Leptakand McKerrow, 1997). TNF-a has been shown to play acentral role in granuloma formation. It restores theability of T cell deficient mice to mount a granulomaaround schistosome eggs (Amiri et al., 1992). Furthersupport for the role of TNF-a has come from studies inwhich the treatment of immunocompetent mice withanti-TNF-a serum resulted in reduced granuloma size(Joseph and Boros, 1993). The present investigationshowed that curcumin treatment reduces granulomavolume and decreases TNF-a serum level. TNF-a isimportant for initiation of granuloma formationthrough upregulation of intracellular adhesion molecule1 (ICAM-1), which mediates cell–cell interactionsand migration across the endothelium (Ritter andMcKerrow, 1996; Brunet et al., 1998). Curcumin wasshown to block TNF-a-induced expression of leukocyteadhesion molecules, including ICAM-1, vascular celladhesion molecule-1 (VCAM-1), endothelial leukocyteadhesion molecule-1 (ELAM-1), and E-selectin (Kumaret al., 1998; Gupta and Ghosh, 1999). Moreover,curcumin has found to inhibit the expression of bothIL-2 (Ranjan et al., 2004; Yadav et al., 2005; Siddiquiet al., 2006) and IL-4 (Kobayashi et al., 1997; Sharmaet al., 2007) in other experimental models. Indeed, bothIL-2 and IL-4 play a role in granuloma formation(Wynn et al., 1993; Cheever et al., 1994; Brunet et al.,1998). Accordingly, curcumin treatment may inhibitgranuloma formation through regulation of cytokinesexpression, especially TNF-a.
Antigens released from eggs trapped in the liverinduce granulomatous inflammatory reactions, whichactivate hepatic stellate cells (HSCs). Upon activation,HSCs proliferate and transform into myofibroblast-likecells that deposit large amounts of connective tissuecomponents, including collagens I and III, and alpha-smooth muscle actin. The predominant extracellularmatrix (ECM) protein synthesized by the HSCs iscollagen type I. Synthesis and secretion of collagen typeI by activated HSCs play a major role in the pathogen-esis of hepatic fibrosis (Karsenty and Park, 1995;Chatterjee et al., 2002). The data of the presentinvestigation showed 38.6% reduction in liver collagencontented of infected treated mice. This result wasconfirmed by our histopathological study that showed adecrease in invasion of the collagen fibres in the liversections of infected treated mice. This finding could beattributed in a part to the decrease of worm and eggburdens as well as granuloma volume with curcumintreatment. It has been estimated that one of the factorsknown to affect the collagen deposition and develop-ment of periportal fibrosis is the parasite load (Cheever,
ARTICLE IN PRESSG. Allam / Immunobiology 214 (2009) 712–727722
1969). Fibrosis following granulomatous inflammationhas also been shown to be dependent on the fibrogenicaction of cytokines such as IL-4, TGF-b and IL-13(Czaja et al., 1989; Cheever et al., 1994; Kaviratne et al.,2004). Curcumin has been shown to suppress IL-4expression (Kobayashi et al., 1997; Sharma et al., 2007)and inhibits the TGF-b induced synthesis of fibronectinin mouse epidermal keratinocytes (Santibanez et al.,2000). In other experimental models, curcumin has beenshown to decrease collagen mRNA expression by 50%(Leclercq et al., 2004). Curcumin’s protective effectagainst liver fibrosis may be associated with its ability toinhibit NF-kB in Kupffer cells and infiltrating macro-phages and consequently inhibit HSCs activation (Brucket al., 2007). Suppression of HSCs proliferation andactivation of ECM biosynthesis have been proposed astherapeutic strategies for the treatment and preventionof the hepatic fibrosis that leads to irreversible livercirrhosis (Duvoix et al., 2005).
The present study clearly showed that S. mansoni
infection leads to a significant decrease of ALT, AST,g-GT, and LDH activities in the liver tissue homo-genate. This depletion in liver enzymes activities couldbe due to leakage of these enzymes from necrotic tissueand the replacement of normal liver tissue by granulo-matous lesions and fibrosis (Allam and Ahmed, 2005;Allam, 2007). However, infected mice treated withcurcumin restore the hepatic ALT, AST, g-GT, andLDH activities that it was decreased by S. mansoni
infection. This amelioration in liver enzymes activitiescould be attributed to the reduction in hepatic granu-loma size and fibrosis as well as absence of necrotic livertissue in infected treated mice. On the other hand, theactivity of anti-oxidant enzyme, catalase, in the livetissue of infected mice also increases with curcumintreatment. Curcumin has been found to induce and/oractivate a number of hepatic antioxidant enzymes,including catalase, superoxide dismutase and the glu-tathione system (Sharma et al., 2001; Iqbal et al., 2003;Joe et al., 2004). Catalase detoxifies hydrogen peroxideproduced by inflammatory cells to water (Inoue, 1994).Therefore, curcumin may protect hepatocytes fromdamage, demise, and dysfunction that caused byoxidative stress at the sites of inflammation.
It is of interest to note that curcumin treatmentdecreases both liver and spleen weight of infected mice.This alleviating effect on Schistosoma-induced hepato-spleenomegaly could be attributed to the reduction ofportal inflammation, granuloma volume, cellular hyper-plasia and fibrosis accompanied with curcumin treat-ment. Such amelioration in schistosomiasis pathologywas reflected on body weight of infected treated micewhich become non-significantly different than that ofnormal mice. Regarding to the effect of curcumintreatment on circulating peripheral blood leukocytes,the present study demonstrate that infected treated mice
showed no significant alteration in total leukocytecount, neutrophil, or moncocyte count in comparisonwith IU one. As expected, eosinophil and neutrophilpercentage were increased, but lymphocyte count wasdecreased with S. mansoni infection. This finding agreeswith previous studies showing a significant increase inboth eosinophil and neutrophil, as well as a decrease inlymphocyte percentage in mice during acute schistoso-miasis mansoni (Sher et al., 1990; Osman et al., 1997).Curcumin treatment decreases eosinophilia of infectedmice to the normal level. This result may be attributedto the inhibitory effect of curcumin on both IL-5 andGM-CSF production (Kobayashi et al., 1997). BothIL-5 and GM-CSF have been shown to have eosino-philopeiosis activity (Sher et al., 1990; Secor et al.,1990).
In conclusion, this study may provide information forraising the interesting possibility that curcumin mayeventually have therapeutic potential in the treatment orprevention of disorders involving S. mansoni infection.Therefore, the combination of curcumin with one ofanti-schistosome drugs may be the aim for the futurework in this field to evaluate the therapeutic possibilityof such combination.
