Unidade de Leishmanioses, Centro Malária Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugal
Received 13 March 2006; received in revised form 8 September 2006; accepted 12 September 2006Available online 7 November 2006
1. Introduction ment of T-helper-2 (Th2) cells, resulting in IL-4 and IL-10
The genus Leishmania includes around 30 taxa thatinfect mammals, causing various types of disease. Humanleishmaniasis has a wide clinical spectrum, from the natu-rally healing localised cutaneous lesion to the potentiallyfatal visceral leishmaniasis, in which parasites disseminatefrom the site of infection and invade organs and tissues ofthe mononuclear phagocytic system (Moll, 1993). In caninevisceral leishmaniasis, it was proven a strong associationbetween the number of parasites, the severity of the clinicalsigns, and the intensity of the immune response (Campinoet al., 2000). In murine models of cutaneous leishmaniasis, itis widely accepted that resistance to the disease is correlatedwith expansion of T-helper-1 (Th1) cells and production ofIFN-�, while susceptibility is associated with the develop-
production (Heinzel et al., 1989; Gumy et al., 2004). How-ever, this Th1/Th2 clear-cut role has not been evident in vis-ceral leishmaniasis (Honore et al., 1998).
The Th1/Th2 balance deWnes diVerent immune pathwaysthat aVect most, if not all, cells of the immune system, con-trolling the parasite burden observed at the diVerent organsof Leishmania infected mice and the pattern of cytokineproduction can inXuence the susceptibility or resistance toinfection. The main goal of the present study was to charac-terise the course of infection and immunological responsesdeveloped in organs and tissues of the mononuclear phago-cytic system of L. infantum infected BALB/c mice.
2. Materials and methods
2.1. Mice and infection
Female BALB/c mice were purchased from HarlamInterfauna Ibérica SL (Barcelona, Spain) and housed at the
N. Rolão et al. / Experimental Parasitology 115 (2007) 270–276 271
Instituto de Higiene e Medicina Tropical (Lisbon), understable climatic and dietary conditions. In each of the threeexperiments performed, 45 female BALB/c mice with 4–6weeks of age were inoculated by i.p. route with 107 promas-tigotes of L. infantum MON-1 (MCAN/PT/94/IMT205) in0.1 ml of saline solution (Group I). Mice from the ControlGroup (Group C) were inoculated with 0.1 ml of salinesolution only. Animal manipulation was conducted accord-ing to the International Guiding Principles for BiomedicalResearch Involving Animals, as issued by the Council forthe International Organizations of Medical Sciences.
2.2. Sample collection
Five mice from Group I and three mice from Group Cwere sacriWced at days 1, 3, 7, 14, 28, 42, 56, 70 and 84 post-inoculation (pi). At each time point, biological samples(spleen, liver and peripheral blood) were “pooled”, accord-ing to each group. The samples were pooled in order toachieve the optimal lymphocyte concentration to use incytokine assays.
2.3. Parasite load
Parasite load determination was accessed by Real-timeTaqMan® PCR, as previously described (Rolão et al.,2004). BrieXy, 10 mg of spleen and liver from each sacriWcedmouse were pooled according to each group (50 mg of eachorgan per group). Biological samples were then processedfor DNA extraction (PCR-template Preparation kit, RocheDiagnostics GmbH, Mannheim, Germany) and quantiWca-tion (GeneQuant, Amersham Biosciences, Buckingham-shire, United Kingdom). All DNA samples were ampliWedfor parasite load determination.
Mass cultures of L. infantum promastigotes spiked withmouse DNA were used to construct the standard curve.Parasites were counted using a Neubauer hemacytometer(mean value of 10 counts) and cultures were diluted inseries of 10-fold dilutions ranging from 105 to 1 parasite.The diluted parasite cultures were then processed for DNAextraction, as above (PCR-template Preparation kit,Roche) and mixed with DNA from healthy mice.
Primers and probe were designed from a kinetoplastidDNA (kDNA) minicircle sequence of L. infantum (Gene-bank A/N AF169140). DNA samples were analysed usingthe following oligos (Applied Biosystems, Foster City,California): primers LshNRf (forward, 5�-GGTTAGCCGATGGTGGTCTT-3�), LshNRr (reverse, 5�-GCTATATCATATGTCCAAGCACTTACCT-3�) and TaqMan®
internal probe LshNRp (5�-ACCACCTAAGGTCAACCC-3�). PCRs were performed in the ABI PRISM® 5700System (Perkin-Elmer, Applied Biosystems). Two microli-tres of each DNA sample were added to a reaction mixconsisting of 10 �l of TaqMan® Universal PCR MasterMix, No AmpErase® UNG (Applied Biosystems) and 1 �lof unlabeled primers and TaqMan® MGB probe (FAM™dye-labelled) mix (Applied Biosystems), in a Wnal volume
of 20 �l. All samples were performed in duplicate andoptimal conditions for PCR ampliWcation were: 95 °C for10 minutes (min) and 40 cycles of 95 °C for 15 seconds (s),and 60 °C for 60 s. The threshold of detection was auto-matically set at 10 times the standard deviation above themean of baseline emission, representing the backgroundlevel calculated from cycles 6 to 15. To overcome possiblequantiWcation errors due to variations in tissue weighing,results were normalized to the DNA concentration of thesamples, as described elsewhere (Rolão et al., 2004).
2.4. Immune response
Spleen and liver samples were homogenized and pro-cessed for mononuclear cell separation by means of Ficoll(Maluish and Strong, 1986) and Percoll (Goossens et al.,1990) gradients, respectively. One hundred and Wftymicrolitres of cell suspension (2£ 106/ml) was added toeach well of micro-ELISA U-bottom plates and left aloneor incubated with crude Leishmania antigen (10 �g/ml perwell), in a Wnal volume of 200 �l/well. After incubation ina humidiWed chamber for 96 hours (h) at 37 °C/5% CO2,supernatants were collected and processed for cytokinequantiWcation by “sandwich” ELISA (BD Pharmingen,San Diego, USA), namely IFN-�, IL-12, IL-4, IL-10 andTGF-�.
Peripheral blood samples were centrifuged at 425g for15 min at room temperature. Sera were removed and pro-cessed for IgG1 and IgG2a detection by ELISA (CaltagLaboratories, Burlingame, USA).
2.5. Statistical analysis
The correlation between the kinetics of parasite load andcytokine production was determined by Spearman’s rankcorrelation analysis.
3. Results
Presented results are representative of the three experi-ments performed.
3.1. Parasite load
Leishmania DNA was detected in all time-points afterinoculation in infected group (Fig. 1). Parasite load reachedhigher values in spleen than in liver. It peaked at days 7 and56 pi (maximum) in the spleen, and reached a maximum atday 84 pi in the liver. Parasite DNA was not detected onsamples from Group C.
PCR eYciency of ampliWcation was 95.82% and theinter-assay coeYcient of variance, calculated from four rep-licates of the same 10-fold DNA dilutions performed onseparated runs, was de 1.13, 2.37, 4.09, 0.27, 0.91, and 1.89%for 105, 104, 103, 102, 10, and 1 parasite, respectively. Intra-assay coeYcient of variation, calculated from a single runof four duplicated 10-fold dilution samples on the same
272 N. Rolão et al. / Experimental Parasitology 115 (2007) 270–276
plate, was 2.03, 1.36, 0.47, 0.81, 0.45, and 0.58% for 105, 104,103, 102, 10, and 1 parasite, respectively.
3.2. Immune response
In Group I, the cytokine kinetics in spleen (Fig. 2) andliver (Fig. 3) was generally associated with the oscillationsof parasite load values. This was particularly evident innon-stimulated cells, where positive correlation betweenparasite load and cytokine production was signiWcant
Fig. 1. Parasite load in spleen and liver of L. infantum infectedBALB/c mice (Group I).
Group I
1,E-01
1,E+00
1,E+01
1,E+02
1,E+03
1,E+04
1,E+05
1,E+06
1,E+07
0 20 40 60 80 100
Days pi
Par
asit
e lo
ad (
log)
(*P < 0.05) for all cytokines except IL-12 in spleen, and forIFN-� and IL-4 in liver. TGF-� was the cytokine that moststrongly correlated with the course of parasite load in non-stimulated cells (**P < 0.01), in which the highest values ofthis cytokine matched the peaks of parasite load in spleenand liver. All cytokines presented similar kinetics, particu-larly in non-stimulated cells, reaching a maximum at day70 pi, when the parasite load was decreased in the spleenand increased in the liver. On day 28 pi, cytokine produc-tion was very low or absent in both organs, with or withoutstimulation. This “immune silence” on day 28 pi wasobserved in the three experiments performed. The kineticsof IFN-� was positively correlated with both IL-4 and IL-10 in all analysed cells (*P < 0.05). Additionally, the highestlevels of cytokine production from both Th1 and Th2 typeswere obtained after day 42 pi, both in spleen and liver.However, the levels of Th2 cytokines IL-4 and IL-10 werehigher in spleen than in liver, while larger amounts of IL-12and TGF-� were detected in liver. Overall, it was notobserved a distinct Th1 or Th2 pattern of cytokine produc-tion during the time of experiment.
Animals from group C produced residual levels of allanalysed cytokines.
The production curves of IgG1 and IgG2a (Fig. 4) werestrongly correlated (**P < 0.01). Both immunoglobulineisotypes started to increase from day 28 pi and then peaked
Fig. 2. Group I (Infected): parasite load and “ex vivo” cytokine production by spleen cells alone or stimulated with 10 �g/ml of Leishmania crudeantigen .
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at days 56 and 84 pi, corresponding to the higher values ofparasite load in spleen and liver, respectively, and whenhigher levels of all cytokines were observed.
4. Discussion
In the present study, Leishmania parasites were detectedin the spleen and in the liver as soon as day 1 pi, and the par-asitism was sustained until the end of the experiment. Melbyet al. (1998) also found parasites in spleen and lymph nodesof L. chagasi (L. infantum synonymous species) infectedmice as soon as 48 h after inoculation. DiVerent workson visceral leishmaniasis experimental infections haveobserved clearance of parasites in the liver of infected micearound 4 weeks pi (Engwerda et al., 1998; Wilson et al.,
Fig. 4. IgG1 and IgG2a production by mice from Groups I(infected) and C (control).
Group C
0,00
0,20
0,40
0,60
0,80
1,00
1 3 7 14 28 42 56 70 84
Days pi
O.D
.
Group I
0,00
0,20
0,40
0,60
0,80
1,00
1 3 7 14 28 42 56 70 84
Days pi
O.D
.
1998; Rousseau et al., 2001). Those diVerences may be dueto variations in virulence between diVerent Leishmania spe-cies (L. donovani and L. infantum/chagasi), diVerent strainsof the same species, or even diVerent clones of the samestrain (Garin et al., 2001; Mendez et al., 2001). However, itshould not be excluded the possibility that the absence ofparasites could be due to the lower sensitivity of the methodused by those authors (impression smears) when comparedto the real-time PCR assay used in the present work. Ourresults are in keeping with other authors that, using a moresensitive method for parasite load determination (limitingdilution assay) found parasites in all spleen and liversamples after two (Melby et al., 2001) or three months post-infection (Honore et al., 1998). Altogether, our resultsdemonstrate the precocity of parasite dissemination to theinternal organs and its competence to establish and main-tain infection in BALB/c mice. Interestingly, in anotherstudy that we performed (data not shown), the inoculationof just 103 promastigotes per mice gave similar results, i.e.,parasite DNA was detected by real-time PCR in all samples,from day 1 to day 84 pi, though with lower values than inmice inoculated with 107 promastigotes. Development ofinfection with a low number of inoculated parasites (103)has also been observed by others using L. major infectedBALB/c mice (Compton and Farrell, 2002) and L. infantuminfected hamsters (Requena et al., 2000).
Fig. 3. Group I (Infected): parasite load and “ex vivo” cytokine production by liver cells alone or stimulated with 10 �g/ml of Leishmania crude
antigen .
274 N. Rolão et al. / Experimental Parasitology 115 (2007) 270–276
From our results, the cytokine kinetics seems to be asso-ciated with variations in parasite load. In spleen, high val-ues of IFN-� are coincident with reductions in parasiteload, suggesting that this cytokine is responsible for theelimination of parasites, as reported by others (Kaye et al.,1991), although it may not be suYcient. It is known thatIFN-� is a potent activator of macrophages for phagocyto-sis and parasite destruction required to the control of anyinfection by Leishmania (Bogdan et al., 1990; Farrell, 2002).Such correlation was not observed in liver, as the high pro-duction of IFN-� on days 56 and 70 pi did not result inreduction of parasite load. One of the reasons that couldexplain the diVerence between both organs may be thehigher level of TGF-� detected in liver. As reported by oth-ers (Vodovotz et al., 1993; Stenger et al., 1994; Goreliket al., 2002; Gantt et al., 2003), TGF-� suppresses theexpression of inducible NO synthase and IFN-� in macro-phages.
In both organs, the kinetics of IFN-� was identical toIL-10 and IL-4, but not to TGF-�. In murine model ofcutaneous leishmaniasis, a paradigm was established link-ing IFN-� to resistance and IL-4 to susceptibility (Locksleyet al., 1987; Scott et al., 1989). However, most studies onexperimental visceral leishmaniasis question about the roleof IL-4 in susceptibility to infection due to the contradic-tory results reported (Kaye et al., 1991; Saha et al., 1993;Miralles et al., 1994). Our results point out to a less signiW-cant role for IL-4 in this visceral model, because, unlikeIFN-� and TGF-�, IL-4 kinetics was not strongly corre-lated with parasite load development. Lehmann et al.(2000) also concluded that the capacity to produce IFN-�rather than the presence of IL-4 determines the eYcacy ofthe immune response in BALB/c mice. Moreover, the roleof IL-4 as a cytokine related to susceptibility has beenrecently questioned even in the experimental cutaneousleishmaniasis model (Sacks and Noben-Trauth, 2002).According to several authors, the anti-inXammatory cyto-kines IL-10 and TGF-� contribute for the establishment ofinfection, as macrophages infected with Leishmania tend toincrease the production of those cytokines, what conducesto a decrease on the leishmanicidal activity (Barral et al.,1993; Bogdan and Nathan, 1993; Stenger et al., 1994). How-ever, it was observed a positive correlation between IL-10and IFN-� production in the present study, which may berelated to a feed-back control. In fact, although promotingsurvival of the parasites, IL-10 is also important for themaintenance of the immunological homeostasis by activa-tion of immune regulatory pathways that may circumscribethe adverse eVects of an exacerbated immunity mediated byTh1 cells (Abbas et al., 1996; Belkaid et al., 2002). Wilsonet al. (1998) also observed that immunization of BALB/cmice upon infection with L. chagasi led to an increased pro-duction of both IFN-� and IL-10. The co-existence of thosecytokines has also been demonstrated in human visceralleishmaniasis (Karp et al., 1993; Kenney et al., 1998; Kempet al., 1999). On the other hand, TGF-� was the cytokinethat most tightly correlated with parasitism oscillations
suggesting a crucial role for this cytokine in favouring para-site multiplication, probably through deactivation of mac-rophage leishmanicidal activity and inhibition of thedevelopment of a Th1-type immune response. Similarresults were reported by Gomes-Pereira et al. (2004). Theseauthors observed that upon Leishmania inoculation of miceof “cure” and “non-cure” phenotypes with L. infantum, theincrease in parasite load in the liver was associated withhigh levels of TGF-�, low levels of a Th2-type cytokinesand lack of development of Th1-type immune response. Inaddition, our results show that the peaks of TGF-� produc-tion often preceded those of the other analysed cytokines,suggesting that TGF-� may trigger immune pathways thatcontrol the production of all other cytokines, playing animportant immunoregulatory role in experimental L. infan-tum infection, as suggested by others (Wilson et al., 1998;Gomes-Pereira et al., 2004), and also reported for otherparasitic diseases (reviewed in Fitzpatrick and Bielefeldt-Ohmann, 1999). TGF-� might be important in maintainingthe balance between control and clearance of infectiousorganisms on one hand and prevention of immune-medi-ated pathology on the other, being its eVects dependent onits concentration at the site of infection or the time it is pro-duced during infection (Omer et al., 2000; Gantt et al.,2003).
The immune “silent phase” observed at day 28 pi may bedue to the presence of other molecules or cells not analysedin this study. Activation of CD4(+)CD25(+) regulatory Tcells may play a role in this suppression, due to their capac-ity to control the immune response (Belkaid, 2003; Sakagu-chi, 2004). More recently, Rodrigues Roos et al. (2005)observed a peak of expression of foxp3, a transcription fac-tor that plays a critical role in the development and func-tion of those cells, at day 28 pi in L. infantum infected mice.
The peaks of production of IgG1 and IgG2a were coinci-dent with the peaks of parasite load in spleen (day 56 pi) andliver (day 84 pi) and the kinetics of both IgG isotypes weretightly correlated. Taking into account that IgG1 and IgG2aare associated with the development of Th2 and Th1 immuneresponses, respectively (Liew and O’Donnell, 1993), ourresults point out to the development of a mixed Th1/Th2immune response by L. infantum infected BALB/c mice, con-Wrming the results obtained with cytokine analysis.
In conclusion, the Th1/Th2 bipolarised immuneresponses, thoroughly described in the murine model of L.major experimental infections (Scott, 1998; Rogers et al.,2002), were not observed in this visceral leishmaniasismodel. The infected mice developed a mixed immuneresponse, with concomitant production of IFN-�, IL-4 andIL-10, both in spleen and liver, and both immunoglobulinG isotypes, IgG1 and IgG2a. The immune responses devel-oped in both organs were not eVective in the resolution ofthe infection until the end of the observation period, sug-gesting that, as pointed out by others (Honore et al., 1998;Melby et al., 2001; Marques et al., 2005), the Th1/Th2 bal-ance may not be enough to explain the susceptibility orresistance to L. infantum infection in the BALB/c model,
N. Rolão et al. / Experimental Parasitology 115 (2007) 270–276 275
contrary to the L. major infection. However, as observed inprevious experiments of murine experimental visceral leish-maniasis (Engwerda et al., 1998; Wilson et al., 1998), ourresults suggest that the spleen is more susceptible to L.infantum: higher values of parasite load were detected in thespleen, along with higher levels of IL-4 and IL-10, whileliver cells produced larger amounts of IFN-� and IL-12.Finally, we believe that the important role of TGF-� in theoutcome of L. infantum infection should be investigatedfurther, particularly in natural infections.
Acknowledgments
We acknowledge O. Rodrigues-Roos for the technicalsupport and the technicians J. Ramada and J.M. Cristóvãofor their assistance. This work was supported by FEDER,FCT Project POCTI/CVT/35263/99.
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