Pregnancy enhances the innate immune responsein experimental cutaneous leishmaniasis throughhormone-modulated nitric oxide production
Yaneth Osorio,*,†,‡,1 Diana L. Bonilla,† Alex G. Peniche,† Peter C. Melby,*,‡
and Bruno L. Travi*,†,‡,1,2
*Department of Medicine, University of Texas, Health Science Center, San Antonio, Texas, USA; †CentroInternacional de Entrenamiento e Investigaciones Medicas-CIDEIM, Cali, Colombia; and ‡Research Service,Department of Veterans Affairs Medical Center, South Texas Veterans Health Care System, San Antonio, Texas, USA
Abstract: The maintenance of host defense dur-ing pregnancy may depend on heightened innateimmunity. We evaluated the immune responseof pregnant hamsters during early infectionwith Leishmania (Viannia) panamensis, a causeof American cutaneous leishmaniasis. At 7 dayspost-infection, pregnant animals showed a lowerparasite burden compared with nonpregnantcontrols at the cutaneous infection site (P�0.0098) and draining lymph node (P�0.02).Resident peritoneal macrophages and neutro-phils from pregnant animals had enhanced Leish-mania killing capacity compared with nonpreg-nant controls (P�0.018 each). This enhancedresistance during pregnancy was associated withincreased expression of inducible NO synthase(iNOS) mRNA in lymph node cells (P�0.02) andhigher NO production by neutrophils (P�0.0001). Macrophages from nonpregnant ham-sters infected with L. panamensis released highamounts of NO upon estrogen exposure(P�0.05), and addition of the iNOS inhibitorL-N6-(1-iminoethyl) lysine blocked the inductionof NO production (P�0.02). Infected, nonpreg-nant females treated with estrogen showed ahigher percentage of cells producing NO at theinfection site than controls (P�0.001), whichcorrelated with lower parasite burdens (P�0.036). Cultured macrophages or neutrophilsfrom estrogen-treated hamsters showed signifi-cantly increased NO production and Leishmaniakilling compared with untreated controls. iNOSwas identified as the likely source of estrogen-induced NO in primed and naıve macrophages,as increased transcription was evident by real-time PCR. Thus, the innate defense againstLeishmania infection is heightened during preg-nancy, at least in part as a result of estrogen-mediated up-regulation of iNOS expression andNO production. J. Leukoc. Biol. 83:1413–1422; 2008.
Key Words: Leishmania � hamster � estrogen
INTRODUCTION
During pregnancy, the immune response is modulated to avoiddeleterious effects on the developing fetus. It is generallyaccepted that gestation is associated with diminished cellularimmunity [1–3] with decreased expression of proinflammatorycytokines and NK cell activation, responses that could lead tofetal abortion [4–6]. It is believed that the down-regulation ofadaptive immune responses makes pregnant females moresusceptible to a wide variety of pathogens, most notably Plas-modium spp. [3]. This observation extends to laboratory ani-mals infected with Toxoplasma gondii, Listeria monocytogenes,Mycobacterium leprae, and Leishmania major [7, 8]. Pregnantmice infected with L. major developed more severe diseasethan nonpregnant controls, which was associated with a skewedTh2 response characteristic of the susceptible phenotype [7].Also, a few anecdotal observations in humans indicated thatwomen infected subclinically with Leishmania donovani orLeishmania infantum developed overt disease during gestation[9, 10]. This evidence suggests that pregnancy may increasethe risk of developing leishmaniasis; however, there are noepidemiological data to confirm this suggestion [11].
On the other hand, several studies have suggested thatheightened innate immunity plays a central role in the main-tenance of host defense throughout gestation [1, 12]. Pregnantwomen have a higher number of activated peripheral bloodmonocytes and granulocytes than nonpregnant women [13–15].Innate immune function in leishmaniasis is of particular im-portance, as the effector cells, i.e., macrophages and to someextent, neutrophils, serve as host cells for the parasite [16, 17].However, to our knowledge, there are no studies that addressthe role of these cells in innate host defense against Leishma-nia infection in pregnant humans or experimental animals.
1 Current address: Department of Medicine, University of Texas, HealthScience Center, San Antonio, TX, USA.
2 Correspondence: Department of Medicine, University of Texas, HealthScience Center, 7703 Floyd Curl Dr., Mailcode 7881, San Antonio, TX78229-3900, USA. E-mail: [email protected]
Received February 26, 2007; revised January 11, 2008; accepted January22, 2008.
In the last decades, an increasing body of evidence hasestablished the relationship between sex hormones and theimmune system. Previous studies demonstrated that increasedconcentrations of estrogen led to enhanced macrophage phago-cytic activity [18] and that estrogen as well as progesterone andprolactin stimulated cytotoxic mechanisms through the releaseof reactive oxygen species [19, 20]. The production of NO,which is critical to Leishmania containment in the mousemodel [21], is also enhanced by the high physiological levels ofestrogen and progesterone in pregnant females of differentspecies. In fact, NO is critical for controlling uterine contrac-tility, cervical ripening, and feto-placental blood flow [22–26].In pregnant rats, progesterone seems to act synergistically withNO, playing an important role in embryo implantation [27, 28].
Experimental studies in hamsters infected with L. infantumindicated that females were protected during lactation [29].Furthermore, female mice or hamsters were shown to be moreresistant to cutaneous leishmaniasis (caused by Leishmaniamexicana and Leishmania (Viannia) panamensis, respectively)than males, and this difference depended, at least in hamsters,on the levels of circulating sex hormones [30, 31]. In this study,we explored the impact of pregnancy and its related hormoneson the innate immune response to experimental cutaneousinfection with L. panamensis. For this purpose, we used anestablished hamster model [31, 32] of American cutaneousleishmaniasis and demonstrated through in vitro and in vivoexperiments that early parasite control in pregnancy was asso-ciated with an estrogen-mediated increase in NO production bymacrophages and neutrophils.
MATERIALS AND METHODS
Parasites
A L. panamensis strain (MHOM/COL/84/1099), which is highly pathogenic forhamsters, was used in all experiments. The strain was episomally transfectedby electroporation with a luciferase (LUC) reporter gene in a pGL2�-Neomi-cin-� plasmid as described [33]. Dr. Marc Ouellette (University of Laval,Canada) kindly provided the LUC reporter plasmid. The transfected Leishma-nia was selected and cultured in complete Schneider’s culture medium sup-plemented with 10% FCS, 2 mM glutamine, 100 units/mL penicillin, 100�g/mL streptomycin, and 0.04 mg/mL G418 neomycin. The virulence andpathogenicity of the transfected Leishmania were determined in previousstudies [34]. No significant plasmid loss was observed up to 2 months post-infection (p.i.) in the strain used in the present study.
Parasite burden
Parasite burden in the lesions was determined at 7 days p.i. by luminometry[33] using a modified method as described by Henao et al. [34]. Briefly, thewhole lesion was excised and immediately homogenized by gentle and thor-ough scraping of the dermis using a scalpel; subsequently, the tissue was lysedby incubation for 30 min at room temperature with 200 �L 5� lysis buffer (125mM Tris-HCl, pH 7.8, 10 mM DTT, 50% glycerol, and 5% Triton X-100) andstored at –70°C until processing. After thawing, the lysate was centrifuged at10,000 rpm, and 20 �L of the supernatant was dispensed in an opaque, white96-well plate. Fresh assay buffer (80 �L; 25 mM Tris-HCl buffer, pH 7.8, 2.67mM MgSO4, 0.1 mM EDTA, 0.53 mM ATP, 33.3 mM DTT, 4.7 �M D-luciferin)was added to the plate immediately before reading in a luminometer (Anthos,Austria; 12 wells per reading) using an integration time of 20 s at 37°C and theautomatic addition of 100 �L assay buffer. The number of parasites wasextrapolated from a linear standard curve generated with LUC-transfectedamastigotes [34].
Infection of animals
Outbred adult (4 months old), female hamsters were used in the studies.Animals were maintained according to the Guiding Principles for BiomedicalResearch Involving Animals (Council for International Organizations of Med-ical Sciences), the Colombian Law 84 of 1989 of the “Estatuto Nacional deProteccion de los Animales,” and the Resolution #008430 of 1993, whichcomplements Law 84. These studies were reviewed and approved by theInstitutional Animal Care and Use Committee of the Centro Internacional deEntrenamiento e Investigaciones Medicas (CIDEIM; Columbia). Pregnant ham-sters were obtained after mating with males during two oestral cycles (8 days).Seven days after mating, female hamsters and nonmated control females wereinoculated intradermally in the snout with 104 wild-type or LUC-transfected L.panamensis stationary-phase promastigotes suspended in 0.05 mL PBS. Eval-uation of the infection during pregnancy was carried out at 7 days p.i. orpost-parturition at 30 days p.i. Animals were killed in a CO2 chamber followinganesthesia with 50 mg/Kg ketamine clorhydrate (Ketamina�, Holliday-ScottS.A., Argentina) and 0.02 mg/Kg xylazine (Rompun�, Bayer, Colombia). Inanimals killed prior to parturition, pregnancy was confirmed by visual inspec-tion of the uterus.
Cytokine and inducible NO synthase (iNOS;NOS2) mRNA expression in pregnant hamsters
Cytokine and iNOS mRNA expression was determined ex vivo by RT-PCRusing lesion tissue or draining lymph nodes, cultured with leishmanial antigen(1�106 freeze-thaw, inactivated promastigotes) during 20 h at 37°C, 5% CO2,in RPMI and 5% FCS. Evaluations were made at 7 or 30 days p.i. The primersequences were derived from published sequences [35, 36] and validated foruse in RT-PCR previously [31, 32]. In addition, specific primers for TNF-�(forward 5�-CACAATCCTCTTCTGCCTGC-3� and reverse 5�-TGTCTTT-GAGAGACATCCCG-3�; expected product, 242 bp) were used. RNA extrac-tion and RT were performed in a final volume of 120 �L as described by Osorioet al. [32]. The cDNA (10 �L per sample, equivalent to �200 ng reverse-transcribed RNA) was denatured at 94°C during 2 min and amplified by PCR(94°C 15 s, 55°C 30 s, and 72°C 1 min, with a final 3-min extension at 72°C)for 30 cycles. Less abundant transcripts (IL-4 and IL-12p40) were subjected to35 amplification cycles. iNOS expression was determined by RT-PCR in lesiontissue and cells from draining lymph nodes stimulated with Leishmania anti-gen, using primers specific for hamster iNOS: forward (exon 12) 5�-ACCACA-CAGCCTCCGAGTCC-3� and reverse (exon 13) 5�-CTGCCAGATGTGGGTCT-TCC-3�; expected product, 200 bp. The expression of iNOS and cytokine (IL-4,TNF-�, TGF-�, IFN-�, IL-10, IL-12p40m) mRNA was analyzed by densitom-etry using image analysis (Gel Doc�, Bio-Rad, Hercules, CA, USA) andnormalized to the expression of the hypoxanthine phosphoribosyltransferasegene (HPRT).
Identification of cell populations byflow cytometry
Lymphocytes, mononuclear cells, and granulocytes were defined by the sizeand granularity of cells [forward-scatter/side-scatter (FSC/SSC)] on a FACS-Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA). CD4� Tlymphocytes and B lymphocytes from peripheral blood were identified usingFITC-labeled anti-mouse mAb that cross-react with the corresponding hamstermolecules (GK1.5 and 14-4-4S, PharMingen, San Diego, CA, USA, respec-tively) [37, 38]. Neutrophils were obtained from pooled, EDTA-treated hamsterperipheral blood following a standard precipitation method with Dextran T-500(Pharmacia, Uppsala, Sweden) [39]. Cells were then sorted by size and gran-ularity (SSC and FSC) on a FACSCalibur flow cytometer (Becton Dickinson).All neutrophil preparations had 99% purity confirmed by Giemsa stain anddirect microscopy.
Leishmanicidal activity by flow cytometry
To estimate the proportion of cells containing viable parasites, we infect thecells with promastigotes previously labeled with 1 �M CFSE dye (MolecularProbes, Eugene, OR, USA). After infection (20 min, 1 h, 34°C, 5% CO2),viable CFSE Leishmania were detected by gating in the cell population,excluding small FSC/small SSC extracellular parasites, and analyzing thepositive region of the fluorescence 1 (FL1) channel. Flow cytometry analysis
1414 Journal of Leukocyte Biology Volume 83, June 2008 http://www.jleukbio.org
was accomplished using the CellQuest (BD Biosciences, San Jose, CA, USA)program.
Parasite replication in macrophages
Resident peritoneal macrophages were obtained from pregnant or controlhamsters by peritoneal lavage with RPMI-EDTA medium. To quantify para-sites replicating within macrophages, 106 resident peritoneal macrophageswere distributed in 24-well plates and incubated for 24 h at 37°C, 5% CO2.Adherent macrophages were infected with 5 � 106 LUC-transfected L. pana-mensis promastigotes and cultured at 34°C, 5% CO2, for 2 h. After washingaway extracellular parasites with warm Dulbeco’s PBS, macrophages werecultured for an additional 72 h at 34°C and 5% CO2 to allow Leishmaniareplication. Infected host cells were carefully detached with the plastic plungerof a tuberculin syringe in cold PBS, counted, adjusted to 100,000 cells, andlysed in 5� LUC lysis buffer. The number of intracellular parasites wasdetermined by luminometry as described above.
NO determination
The fluorescent probe 4-amino-5-methylamino-2�,7�-difluorescein (DAF-FM;Molecular Probes) diacetate was also used to detect the proportion of cellsproducing NO production by flow cytometry. The specificity of NO determi-nations was established by using the iNOS-specific inhibitor L-N6-(1-imino-ethyl) lysine (L-NIL; Cayman Chemical Co., Ann Arbor, MI, USA) dihydro-chloride and the peroxynitrite scavenger manganese (III) tetrakis (4-benzoicacid) porphyrin (Calbiochem, San Diego, CA, USA). The percentage of NO-producing cells was determined in peripheral blood neutrophils, residentperitoneal macrophages, and cells derived from the skin at the site of infection(7 days p.i.). Cells from skin were obtained by mechanical dissociation usinga Medimachine� apparatus (BD Biosciences) following the instructions of themanufacturer. In brief, cells (500,000 cells in 100 �L PBS) were incubatedwith 0.5 �M DAF-FM during 30 min at room temperature in dark. The cellswere washed with PBS and incubated for 15 min to complete de-esterificationof the intracellular diacetates. Fluorescence intensity and percentage of gatedcells expressing NO were determined in the FL1 channel (515 nm). DAF-FM-labeled cells from uninfected animals were used to define the threshold for flowcytometry analysis. Total NO production was evaluated in culture supernatantsof 2.5 � 106 peritoneal macrophages stimulated with antigen and 1000 nMestrogen (48 h in 250 �l final volume) using the Griess reaction (CaymanChemical Co.). The specificity of iNOS-derived NO production in the super-natants was determined by the addition of 1 mM L-NIL. A TaqMan assay wasused to identify iNOS induced by estrogen. Briefly, Universal Master Mix(Applied Biosystems, Foster City, CA, USA) was mixed with 150 ng reverse-transcribed RNA (SuperScript II, Invitrogen, Carlsbad, CA, USA), 700 nMeach primer, and 100 nM TaqMan probe (forward 5�-tga gcc act gag ttc tcc taagg-3�; reverse 5�-tcc tat ttc aac tcc aag atg ttc tg-3�; probe 5�-6FAM-cgt gga cacttc ctt tgt ctg tgc tcc-TAMRA-3�) in a final volume of 25 �l. The reference gene�-actin was used as normalizer and detected in a validated multiplex assayusing a VIC-labeled TaqMan probe. The fold increase in iNOS expression wascalculated using nonstimulated macrophages as calibrator and the compar-ative threshold method.
In vitro assays using estrogen or progesterone
To evaluate the influence of estrogen or progesterone on NO production,resident peritoneal macrophages were obtained by peritoneal lavage fromuninfected hamsters or from hamsters at 7 days p.i. with L. panamensis. Formeasurement of hormone-induced NO, we used macrophages but not neutro-phils, as the latter could only be recovered in limited numbers from peripheralblood. Cells were cultured for 5 min at 37°C, 5% CO2, 95% air, with differentconcentrations of 17-� estradiol (10–1000 nM) or the progesterone metabolite,5�-pregnane-3�-ol-20 (pregnane; 1–100 �M, Sigma Chemical Co., St. Louis,MO, USA). In some experiments, the estrogen receptor inhibitor tamoxifen (1�M) was used to antagonize the action of 17-�-estradiol as described [24]. Theestrogen and tamoxifen were dissolved in absolute ethanol, and dilutions madein RPMI, 5% FCS, 2 mM glutamine, 100 units/mL penicillin, and 100 �g/mLstreptomycin. After treatments, cells were cocultured with L. panamensis at34°C, and parasite survival and proportion of cells producing NO were deter-mined by flow cytometry. iNOS expression was quantitated by real-time PCRas described above.
Treatment of hamsters with estrogenand progesterone
Hamsters were treated intramuscularly with 2 �g 17-�-estradiol (Sigma Chem-ical Co.) or 3 �g of the active progesterone metabolite pregnane (SigmaChemical Co.) every 48 h for 14 days. These treatment schedules were shownpreviously to increase plasma levels of these hormones [40]. Protocols ofinfection and in vitro assays were followed as described for pregnant animals.Hamsters were infected intradermally in the snout with L. panamensis asdescribed before on the 7th day of hormone treatment. At 7 days p.i. (14th dayof hormone treatment), the animals were killed, and the parasite burden wasdetermined by luminometry of homogenized tissue harvested from the infectionsite. A group of animals sham-treated with PBS was included as a control.Leishmanicidal activity and percentage of macrophages, neutrophils, and cellsfrom lesions that were producing NO were determined by flow cytometry asabove.
Statistical analysis
Multiple comparisons ANOVA with post-hoc Duncan’s test or Kruskal-Wallistest with Dunn’s procedure were used at a 95% confidence interval using SPSSstandard program for Windows. Single comparisons were performed usingGraphPad InStat software, Version 3.06, as described in each figure.
RESULTS
Pregnancy enhances the control of earlycutaneous infection with L. panamensis
Female hamsters infected with L. panamensis during preg-nancy developed significantly smaller lesions than nonpreg-nant controls during the early phase of infection (Fig. 1A).This phase included the second half of pregnancy, and thedifference in lesion size was evident at the time of parturition(15 days p.i.; P�0.040) and at the completion of lactation (30days p.i.; P�0.038). In animals infected during pregnancy(n�6), the draining lymph node harbored fewer parasites thanin nonpregnant females (n�4; P�0.02; Mann-Whitney U-test;data not shown). In two different experiments, the number ofparasites was determined in lesion tissue using a LUC-trans-fected L. panamensis strain. We found that pregnant animalshad a 2.5-fold lower number of amastigotes in the infection sitethan control animals (P�0.0098; Fig. 1B).
Pregnancy modulates the cellular immuneresponse to early L. panamensis infection
As it is generally accepted that cellular immune function isaltered during pregnancy, we first examined leukocyte popu-lations in L. panamensis-infected pregnant and nonpregnanthamsters. Pregnant, infected hamsters had a lower percentageof peripheral blood lymphocytes than controls (P�0.01); how-ever, there was a tendency toward an increased proportion ofCD4� T cells in pregnant compared with nonpregnant, in-fected females (P�0.07; Table 1). No differences in theproportion of B lymphocytes, mononuclear cells, or granulo-cytes were found among experimental groups (Table 1).
To identify the potential mechanisms for the enhanced re-sistance to early cutaneous infection during pregnancy, we firstexamined the in situ cytokine response at the site of infection.In general, there was greater expression of cytokine mRNAs inthe cutaneous lesion than in the draining lymph node, andcytokine expression was equivalent to or reduced in the preg-
Osorio et al. Pregnancy and innate immunity in cutaneous leishmaniasis 1415
nant animals (Table 2). Transcripts for the proinflammatoryand macrophage-activating cytokines IL-12p40, IFN-�, andTNF-� were reduced at the lesion site (IL-12, TNF-�) ordraining lymph nodes (IFN-�) in pregnant compared withnonpregnant animals (P�0.05). The expression of the anti-inflammatory, macrophage-inactivating cytokine TGF-� wasalso consistently low in the lesion and draining lymph node inpregnant compared with nonpregnant animals (Table 2). Thus,there was a generalized pregnancy-associated reduction in theearly expression of cytokine mRNA in response to L. pana-mensis infection.
Pregnancy enhances the generation of NO andleishmanicidal capacity of macrophages andneutrophils exposed to L. panamensis
Despite the pregnancy-related reduction in cytokine expres-sion at the site of infection, the consistent clinical and para-sitological control of infection and low parasite burden ofpregnant hamsters was associated with high in situ expressionof iNOS (NOS2). Densitometry analysis of RT-PCR productsshowed that iNOS mRNA expression levels in pregnant ani-mals were 3.5- and 1.4-fold higher in infected skin (P�0.07)and draining lymph nodes (P�0.02), respectively, than thoseof infected, nonpregnant controls (Table 2). These observationsprompted us to evaluate the effect of pregnancy on leishmani-cidal capacity and NO production of macrophages and neutro-
phils exposed to L. panamensis. Resident peritoneal macro-phages harvested from infected, pregnant (n�7) and infected,nonpregnant control hamsters (n�6) showed similar phago-cytic capacities determined 20 min after the uptake of CFSE-labeled promastigotes (mean percentage of macrophages withparasites SD�3 10% and 3 6%, respectively). Accord-ingly, the number of parasites determined by luminometry inadherent macrophages at 20 min–1 h p.i. was similar in preg-nant hamsters and nonpregnant controls (mean number ofamastigotes SE�309,773 54,062, n�11; 369,704 80,499,n�14, respectively). However, after 72 h of culture, macro-phages from infected, pregnant hamsters harbored 3.6-foldfewer amastigotes than the corresponding infected, nonpreg-nant controls (P�0.0181; Fig. 2A). Microscopical examina-
Fig. 1. Pregnancy enhances the control ofearly cutaneous infection with L. panamen-sis. (A) Lesion size. Pregnant female(shaded bars) or nonpregnant female (openbars) hamsters were infected in the snoutwith 104 L. panamensis promastigotes, andthe lesion size (mean SD) was determinedafter 15 days p.i. (parturition) and 30 daysp.i. (end of lactation). Individuals infectedduring pregnancy (n�9) had smaller lesionsthan controls (n�12) at both time-points(P�0.04; unpaired t-test, normality testedby Kolmogorov and Smirnov). (B) Parasiteburden. The parasite burden in the snoutfrom pregnant (n�7) and nonpregnant fe-male hamsters (n�11), infected as described above, was determined by luminometry at 7 days p.i. Data are combined from two independent experiments and arepresented as number of amastigotes per lesion (mean SE; P�0.0098; unpaired t-test).
TABLE 1. Phenotype of Peripheral Blood Cells in PregnantHamsters Infected with L. panamensis (7 Days p.i.) Compared with
a Percentage of positive cells determined by flow cytometry. b Statisticallysignificant between control and pregnant groups by Kruskal Wallis test; 95%confidence.
TABLE 2. Cytokine and NO mRNA Expression in the InfectionSite and Cells from Draining Lymph Nodes of Pregnant Hamsters
Infected with L. panamensis (7 Days p.i.) Compared withNonpregnant, Infected Controlsa
a Data are from a single experiment representative of two or three indepen-dent experiments. b RT-PCR results are expressed as a net value with refer-ence to the HPRT gene, which is constitutively expressed. c Control/pregnantratio indicates the ratio of mRNA that is expressed by controls with referenceto pregnant animals. d Statistically significant difference between control andpregnant groups (Mann-Whitney test).
1416 Journal of Leukocyte Biology Volume 83, June 2008 http://www.jleukbio.org
tions confirmed this observation; at 72 h of culture, macro-phages from pregnant females (n�6) harbored 0.9 0.7(mean SD) amastigotes per macrophage, and macrophages ofcontrol females (n�7) had 4.0 4.6 (mean SD) amastigotesper macrophage (P�0.037; Mann-Whitney test).
Leishmanicidal activity was also studied in neutrophils frompregnant animals. The proportion of neutrophils that phagocy-tosed Leishmania was similar in pregnant and control neutro-phils (pregnant�95 2.4, pooled sample, n�9; con-trols�90.3 9.2, pooled sample, n�7), as determined by theproportion of sorted neutrophils containing Leishmania. How-ever, the analysis of intracellular amastigote viability, usingCFSE-labeled parasites, showed that a lower percentage ofneutrophils from pregnant compared with nonpregnant ham-sters contained viable Leishmania (P�0.0187; Fig. 2B).
L. panamensis-triggered NO production by macrophages orneutrophils was determined in a fluorescence-based (DAF-FM)flow cytometry assay. The specificity of the NO detection wasestablished by using the NO inhibitor L-NIL dihydrochloride(Cayman Chemical Co.). No NO production by L. panamensispromastigotes was detected using DAF-FM. Hamster perito-neal macrophages infected in vitro with L. panamensis promas-tigotes and cultured for 24 h with 10–1000 �M L-NIL showeda dose-dependent decrease in NO production (linear regres-sion, P�0.01); NO production by these macrophages wasabolished at 1000 �M L-NIL (data not shown).
There was a trend toward a higher proportion of residentperitoneal macrophages isolated from infected, pregnanthamsters to produce NO compared with macrophages frominfected, nonpregnant animals (mean SE of five experi-ments: pregnant, 44.8 2.8; control, 34.6 5.1; P�0.07).Additionally, the percentage of NO-producing neutrophilswas 6.8-fold greater in L. panamensis-infected, pregnantanimals compared with infected, nonpregnant animals(mean SE of two different experiments: pregnant, 72.8 8.3;control, 10.7 1.5; P�0.0001). Pregnancy also enhancedthe percentage of neutrophils producing NO in uninfectedanimals (P�0.006). Collectively, these data indicate thatneutrophils from pregnant or infected animals show in-creased NO production over controls and that pregnancyamplifies NO production induced by infection.
Estrogen and pregnane enhance macrophage NO
The efficient parasite control observed in pregnant animalsduring the early stages of infection, which was associated withhigh iNOS expression, suggested that reproductive hormonescould play a role in Leishmania containment. Therefore, wedetermined the influence of the pregnancy-associated hormoneestrogen (17-� estradiol) and pregnane (a progesterone metab-olite) on NO production by macrophages upon in vitro infectionwith L. panamensis. Resident peritoneal macrophages isolatedfrom hamsters infected with L. panamensis (7 days p.i.) andrestimulated with Leishmania antigens showed a dose-depen-dent increase in production of NO after ex vivo exposure toincreasing doses of 17-� estradiol (P�0.01; Fig. 3A). Addi-tion of 1 �M tamoxifen (which competitively blocks the estro-gen receptor) to the culture medium before supplementationwith estrogen impaired the hormone-mediated increase in NOproduction (P�0.056; Fig. 3A). Peritoneal macrophages fromL. panamensis-infected hamsters exposed to this hormone for6 h showed a 35-fold increase in iNOS expression over non-stimulated macrophages (P�0.01; Fig. 3B). Tamoxifen inhib-ited iNOS expression induced by estrogen treatment, suggest-ing iNOS expression by a classic, receptor-mediated pathway(P�0.01; Fig. 3B).
In addition, peritoneal macrophages from infected animalsstimulated for 48 h with L. panamensis antigen and estrogenreleased more total NO to culture supernatants compared withmacrophages cultured with antigen alone (P�0.05). Additionof the iNOS inhibitor L-NIL to antigen/estrogen-stimulatedmacrophages blocked the induction of NO production(P�0.02; Fig. 3C).
Naıve macrophages also showed increased iNOS expressionand NO production upon estrogen treatment. The quantifica-tion of iNOS using a TaqMan assay showed that peritonealmacrophages from uninfected hamsters increased iNOS ex-pression fivefold after 16 h exposure to 1000 nM estrogen(nontreated�onefold 0.45; estrogen�5.3-fold 1.86; P�0.018;unpaired t-test). On the other hand, flow cytometry evaluationsshowed that the percentage of macrophages producing NOincreased sixfold after estrogen treatment (nontreated�3.4% 1.1; estrogen-treated�20.7% 12).
Fig. 2. Pregnancy enhances the leish-manicidal capacity of macrophages andneutrophils exposed to L. panamensis. (A)Leishmanicidal capacity of macrophages.Resident peritoneal macrophages isolatedfrom infected (7 days p.i.), pregnant andnonpregnant hamsters were infected in vitrowith L. panamensis, and the parasite burdenwas determined after 72 h by luminometry.Shown is the number of amastigotes(mean SE) obtained in three independentexperiments using four to five hamsters pergroup per experiment (P�0.018; unpairedt-test). (B) Leishmanicidal capacity of neu-trophils (N�s). Sorted peripheral blood neu-
trophils were pooled and infected with CFSE-labeled promastigotes. The percentage of neutrophils harboring viable CFSE � L. panamensis was determined byflow cytometry at 20 min–1 h p.i. Shown is the mean SD percentage of neutrophils containing viable leishmania observed in one experiment that is representativeof two independent experiments. Neutrophils from pregnant hamsters (nine pooled samples) had greater leishmanicidal capacity than neutrophils from thenonpregnant animals (seven pooled samples; P�0.018; unpaired t-test).
Osorio et al. Pregnancy and innate immunity in cutaneous leishmaniasis 1417
Treatment of peritoneal macrophages with concentrations ofpregnane ranging from 1 to 100 �M significantly increased thepercent of NO-producing cells (sixfold) compared with cellsfrom infected or uninfected animals sham-treated with mediumalone (P�0.001; Fig. 3D). An increase in the percentage ofNO-positive cells was not observed at lower doses of pregnane(1–500 nM).
In vivo administration of estrogen and pregnanemodulates host NO production andparasite control
To determine the in vivo relevance of the previous ex vivo andin vitro observations, different groups of hamsters were treatedwith the aforementioned hormones. Estrogen or pregnane wasadministered to hamsters every 48 h for 14 days, and theanimals were infected on Day 7 of hormone treatment withLUC-transfected L. panamensis to determine NO-producingresident peritoneal macrophages and peripheral blood neutro-phils; parasite survival after in vitro infection of residentperitoneal macrophages and peripheral blood neutrophils; andparasite burden and NO producer cells at the infection site byluminometry or flow cytometry, respectively.
Seven days after infection of hamsters that had been treatedwith estrogen or pregnane, we found that NO production byresident peritoneal macrophages was greater in the hormone-treated compared with untreated animals. Resident macro-phages collected from infected hamsters treated with estrogenproduced eightfold more NO than those treated with PBS(P�0.001; Table 3). Also, there was a tendency of macro-phages from pregnane-treated hamsters to secrete higher levelsof NO (threefold; not significant) than controls. Macrophagesfrom estrogen-treated hamsters demonstrated significantlygreater leishmanicidal capacity than controls; this enhance-ment in Leishmania killing ability could not be detected inmacrophages derived from animals treated with pregnane (Ta-ble 3). A significant inverse correlation was found in thepercentage of estrogen-induced, NO-producing cells and par-asite survival in peritoneal macrophages (Spearman’s correla-tion; P�0.041).
In the hamsters treated with estrogen or pregnane, we alsofound that NO production by peripheral blood neutrophils wasthree- to fourfold higher than in neutrophils from infected,control animals that had been sham-treated with PBS (Table 3).In addition, peripheral blood neutrophils from infected animals
Fig. 3. Estrogen and progesterone mod-ulate NO production and iNOS expressionin resident peritoneal macrophages fromhamsters infected with L. panamensis.Resident peritoneal macrophages wereisolated from hamsters infected with L.panamensis and exposed in vitro for 5 minto estrogen (17-� estradiol; 0 –2000 nM),with or without tamoxifen (1 �M) or preg-nane (5�-pregnant-3�-ol-20; 1–100 �M).The estrogen receptor inhibitor tamoxifenwas included 30 min before estrogentreatment and L. panamensis infection.(A) Effect of estrogen (E2) on the propor-tion of NO-producing cells determined byflow cytometry. Estrogen was added to themacrophage cultures that were stimulatedwith L. panamensis promastigotes. Thedata shown (mean SD) are from a singleexperiment representative of two indepen-dent experiments with four hamsters each;the threshold in the experiment was es-tablished using uninfected hamsters(Uninf). The frequency of NO-producingcells increased with estrogen treatment(P�0.01) and decreased by blocking theestrogen receptor with tamoxifen (P�0.056;Kruskal-Wallis test; nonparametric ANOVA).(B) Estrogen-induced iNOS expression(real-time PCR). iNOS mRNA expressionin peritoneal macrophages from hamstersinfected with L. panamensis 6 h after treatment with 1000 nM estrogen or estrogen � 1 �M tamoxifen (TX). Data of four replicates from pooled samplesof five animals are shown as fold increase with respect to cells cultured with medium alone. Estrogen treatment induced iNOS mRNA expression (P�0.01)and was blocked with tamoxifen (P�0.01; unpaired t-test). (C) Effect of estrogen on iNOS-derived NO production. Resident peritoneal macrophages(2.5�106) isolated from L. panamensis-infected female hamsters (n�5) were stimulated for 48 h with L. panamensis (L.p.) antigen (100,000 frozen-thawedL. panamensis promastigotes) in the presence or absence of estrogen (1000 nM), with or without iNOS inhibitor L-NIL (1 mM). NO (mean SD) released inthe supernatants after 48 h of in vitro culture, determined by Griess reaction, showed that estrogen treatment induced NO production (P�0.05), which wasblocked with the addition of tamoxifen (P�0.02; unpaired t-test). (D) Effect of progesterone on NO production. Resident peritoneal macrophages fromhamsters infected with L. panamensis were exposed in vitro to 1–100 �M pregnane, and following 1 h of in vitro coculture with L. panamensis, theNO-producing cells were identified by reaction with DAF-FM diacetate and flow cytometry. The frequency of NO-producing cells was increased withpregnane treatment (P�0.001; Mann-Whitney test).
1418 Journal of Leukocyte Biology Volume 83, June 2008 http://www.jleukbio.org
that had been treated with estrogen showed significantlygreater parasite killing following ex vivo infection than theanimals that did not receive estrogen (P�0.03; Table 3).However, no differences were found in the leishmanicidalcapacity of neutrophils harvested from infected hamsterstreated with pregnane and those collected from infected ani-mals not treated with the hormone (Table 3).
At 7 days p.i., prior to the detection of a measurable cuta-neous lesion, the parasite burden in the inoculation site wassignificantly lower in estrogen-treated hamsters than in con-trols (Fig. 4A; P�0.036). A similar but not significant trendwas observed in animals treated with pregnane (Fig. 4A).Consistent with the in vitro observations, flow cytometry anal-ysis showed that NO levels in the infected skin of hamsterstreated with estrogen were higher than in the correspondingcontrols (P�0.001; Fig. 4B). Also, a negative correlation be-tween the proportion of NO-producer cells and parasite burden
was found in the dermis of hamsters treated with estrogen orpregnane (Spearman’s correlation; P�0.05).
DISCUSSION
The present study is the first to explore the role of phagocytesin the innate immune responses to experimental cutaneousleishmaniasis during pregnancy. The clinical and parasitolog-ical results clearly indicated that during the early stages ofinfection, pregnant female hamsters were capable of control-ling Leishmania more effectively than nonpregnant individuals.This heightened control of Leishmania infection was paralleledby enhanced innate immune responses, as demonstrated byincreased NO generation and parasite killing by macrophagesand neutrophils during the first week of infection. Pregnancyacted additively with cutaneous infection to enhance iNOSmRNA expression and NO production locally (inoculation siteand draining lymph node) and systemically (resident peritonealmacrophages and peripheral blood neutrophils). The adminis-tration of the pregnancy-related hormones estrogen and pro-gesterone to hamsters or to in vitro-cultured macrophagesrecapitulated the increased leishmanicidal activity observed inpregnancy.
The activation of innate immunity during pregnancy, as weobserved in this study in hamsters, has been described inhumans [12, 14, 15]. Furthermore, female hamsters that wereinfected with L. infantum 24 h after parturition were also foundto have less evidence of visceral leishmaniasis and a reducedvisceral parasite burden [29]. The influence of elevated prolactinwas considered to be the cause of the increased resistance;however, our data suggest that other residual, pregnancy-related hormonal changes could have also contributed to theenhanced resistance in this early challenge model. Our find-ings are distinct from what was reported for pregnant C57BL/6mice infected with L. major, in which the latter were moresusceptible than nonpregnant females [7]. This apparent dis-crepancy could be in part a result of differences in the Leish-mania strain used and/or host species between the studies. Thestudy of Krishnan et al. [7] focused principally on the devel-
TABLE 3. Percentage of Cells Producing NO and Percentage ofViable Parasites in Peritoneal Macrophages or Peripheral Blood
Neutrophils from Hamsters Treated with Estrogen (17-�-Estradiol)or Pregnane (5�-Pregnant-3�-ol-20)a
a Data are representative of two experiments using four animals per group.b NO production evaluated using flow cytometry and DAF-FM. c ViableCFSE� parasites measured after the in vitro infection of cells obtained fromfemale hamsters at Day 14 of treatment (7 days p.i. with L. panamensis).d Comparison made between hormone-treated and PBS-treated groups. e K-ruskal-Wallis test with post-hoc Dunn’s correction for multiple comparisons.f ANOVA; Duncan’s post-hoc test. NS, Not significant.
Fig. 4. Hormone treatment modulates par-asite burden and NO production at the siteof infection. Female hamsters (n�6 pergroup) were treated with 2 �g 17-�-estra-diol (Estrogen) in PBS, 3 �g 5�-pregnane-3�-ol-20 (Pregnane) in PBS, or PBS aloneevery 48 h for 14 days. The animals wereinfected with L. panamensis in the snout onDay 7 of treatment. The number of animalsin each group is shown on the horizontalaxis. (A) Parasite burden at the site of in-oculation. The parasite burden in the snoutwas determined 7 days p.i. by luminometry.The data (amastigote numbers) are shown asa box plot (median plus first–third quartile)with whiskers showing the smallest and
largest values. Significant differences were identified between PBS and estrogen treatment (P�0.036) but not between PBS and pregnane (Kruskal-Wallis test andDunn procedure). (B) NO-producing cells at the site of inoculation. NO production by cells in the infected snout tissue was evaluated by flow cytometry ofDAF-FM-labeled cells. Significant differences were identified between PBS and pregnane treatment (P�0.001) and PBS and estrogen (P�0.026; Kruskal-Wallistest; nonparametric ANOVA).
Osorio et al. Pregnancy and innate immunity in cutaneous leishmaniasis 1419
opment of the adaptive, T cell-mediated immune response in achronic infection model rather than in the early events ofinnate immunity as studied here. Nevertheless, our studies donot exclude the possibility that females infected during preg-nancy could ultimately develop a Th2 response that worsenschronic infections.
Parasite internalization by resident peritoneal macrophageswas found to be unaltered during pregnancy, but macrophagesfrom pregnant, infected hamsters had enhanced capacity torestrict Leishmania replication at 72 h p.i. The increasedleismanicidal activity of hamster macrophages and neutrophilsduring pregnancy was accompanied by a general decrease inexpression of cytokines (IFN-�, IL-12, and TNF-�) typicallyassociated with classical macrophage microbicidal activity.This cytokine down-regulation concurs with the classic para-digm of suppression of T cell responses (without a Th2 cytokinebias) during human pregnancy [1–3, 13–15] but differs from aprevious report of an enhanced Th2 (IL-4) response in pregnantmice infected with L. major [7]. The relatively greater leish-manicidal activity of macrophages from pregnant females, de-spite the reduced expression of classical macrophage activa-tors, may be related to the reduced expression of TGF-� and toa lesser extent, IL-10, cytokines known to suppress macro-phage effector function and promote Leishmania infection [41].Alternatively, macrophages of pregnant females might haveenhanced responsiveness to IFN-� through up-regulation ofexpression of IFN-� receptors, as has been described in myo-metrial macrophages of pregnant mice [42].
Our results indicate that during the first days of infection,not only macrophages but also neutrophils from pregnant fe-males generated more NO and killed Leishmania more effec-tively than those from nonpregnant animals. The leishmani-cidal capacity of neutrophils has also been demonstrated inhuman cells infected ex vivo with L. major [43] in resistantmouse strains [44] and in hamsters infected with L. panamensis(unpublished), suggesting that they constitute an early, primarybarrier to Leishmania infection. However, other reports havesuggested that neutrophils could also act as a “vector” forintracellular Leishmania via a parasite-induced delay in apo-ptosis [16, 43, 45] or through the phagocytosis of apoptotic,amastigote-laden neutrophils by human macrophages. Our dataindicate that during pregnancy, the neutrophils were highlyactivated and had enhanced parasite killing, so were probablynot acting as permissive Leishmania host cells.
Hamsters, like humans, express iNOS at levels substantiallylower than mice [36, 46]. We demonstrated previously that inL. donovani-infected hamsters and in IFN-�/LPS-stimulatedhamster macrophages, iNOS mRNA expression and NO pro-duction could not be detected by Northern blot and Greissreaction, respectively [36, 46]. The data presented here are notincongruent with these previous findings for several reasons.First, NO-producing macrophages and neutrophils were de-tected in this hamster model of cutaneous L. panamensis in-fection using a much more sensitive, fluorescence-based flowcytometry assay, and similarly low levels of NO production andiNOS were measured by Griess reaction and real-time PCR.Second, parasite-specific differences in the induction of iNOS
expression are evident; the relatively controlled, cutaneous L.panamensis infection is accompanied by iNOS expression,whereas the progressive, fatal L. donovani infection actually iscoincident with suppressed iNOS expression (our unpublishedobservations). That the generation of reactive nitrogen speciesis stimulus-specific is further supported by the demonstrationof iNOS expression and NO production in hamsters immunizedagainst L. donovani [47] and in hamsters infected with Enta-moeba histolytica [48].
This work adds support to a growing number of reports, inexperimental animals and humans, that estrogen is importantfor iNOS expression and NO production by different cell types,including monocytes and macrophages [24, 26, 49]. The exvivo and in vitro studies presented here identify a direct effectof estrogen on iNOS in macrophages, which was mediatedthrough the estrogen receptor (our data and refs. [25, 26]).
We confirmed that the estrogen-enhanced NO productionand iNOS expression found in cultured phagocytes were alsoevident in vivo following administration of estrogen to nonpreg-nant hamsters. Most notably, the estrogen-enhanced phagocyteleishmanicidal activity was associated with improved control ofthe early phase of cutaneous infection. In addition to the directeffect on macrophages, at the site of infection in vivo, theestrogen effect might be mediated by T cell-dependent signals[50, 51].
Physiological studies have also associated the increasedprogesterone levels in pregnancy with the biosynthesis of NO[27, 28, 40]. The administration of pregnane to hamsters in-creased the number of NO-producing cells at the site of cuta-neous infection and by ex vivo-cultured macrophages andneutrophils. However, there was no pregnane-mediated in-crease in parasite killing in the lesion or by ex vivo-culturedmacrophages or neutrophils. This suggests that a hormone-mediated increase in NO production is not sufficient in and ofitself to affect parasite killing and that there must be otherestrogen-induced effector mechanisms that act additively orsynergistically with the generation of NO.
This experimental study demonstrates that the innate im-mune response, characterized by phagocyte NO production andcontrol of L. panamensis infection, is enhanced during preg-nancy. Although ex vivo studies demonstrated that macro-phages and neutrophils could be a source of the increased NOproduction, and both are found at the site of infection [32],further studies are needed to define more clearly the relativecontribution of these cells to the pregnancy-enhanced NOproduction and parasite killing in vivo. To our knowledge, thisis the first study in which a clear association between estrogen-induced iNOS expression/NO production and Leishmania kill-ing has been found. Significantly, this pregnancy- and hor-mone-enhanced innate host defense was demonstrated in ananimal model whose macrophages show relatively low iNOSexpression and NO production in response to inflammatorystimuli, a feature similar to humans but distinct from themurine rodents. These results underscore the importance ofhormones in the regulation of the immune response and sup-port the concept that physiological changes that are associatedwith varying levels of circulating hormones can impact the
1420 Journal of Leukocyte Biology Volume 83, June 2008 http://www.jleukbio.org
balance between host and pathogens such as Leishmania.Additional studies will need to explore the consequences ofpregnancy on the evolution of the adaptive immune responseand chronic disease. It will also be important to understandhow infection before or during pregnancy in humans couldaffect the natural history of disease in the mother and the child.The knowledge of mechanisms involved in the estrogen-in-duced production of NO is of potential interest for the controlof intracellular pathogens.
ACKNOWLEDGMENTS
This work was supported by the National Program of Scienceand Technology in Health (Colombia; COLCIENCIAS),Projects 22290412603 and 22290414328, and the NationalInstitutes of Health (Bethesda, MD, USA), grant AI 061624 toP. C. M. We thank Dr. Mark Ouellete (University of Laval,Canada) and Dr. John Walker (CIDEIM) for providing theplasmid and transfection of the Leishmania panamensis strainused in this study. We specially thank Osibar Jamauca for thehamster husbandry. The laboratory assistance of Carlos A.Hernandez and Hector Henao and the statistical support ofMauricio Perez are gratefully acknowledged.
2. Jiang, S. P., Vacchio, M. S. (1998) Multiple mechanisms of peripheral Tcell tolerance to the fetal “allograft”. J. Immunol. 160, 3086–3090.
3. Weinberg, E. D. (1984) Pregnancy-associated depression of cell-mediatedimmunity. Rev. Infect. Dis. 6, 814–831.
4. Kelemen, K., Paldi, A., Tinneberg, H., Torok, A., Szekeres-Bartho, J.(1998) Early recognition of pregnancy by the maternal immune system.Am. J. Reprod. Immunol. 39, 351–355.
5. Szereday, L., Varga, P., Szekeres-Bartho, J. (1997) Cytokine production bylymphocytes in pregnancy. Am. J. Reprod. Immunol. 38, 418–422.
6. Wegmann, T. G., Lin, H., Guilbert, L., Mosmann, T. R. (1993) Bidirec-tional cytokine interactions in the maternal-fetal relationship: is success-ful pregnancy a TH2 phenomenon? Immunol. Today 14, 353–356.
7. Krishnan, L., Guilbert, L. J., Russell, A. S., Wegmann, T. G., Mosmann,T. R., Belosevic, M. (1996) Pregnancy impairs resistance of C57BL/6 miceto Leishmania major infection and causes decreased antigen-specificIFN-� response and increased production of T helper 2 cytokines. J. Im-munol. 156, 644–652.
8. Luft, B. J., Remington, J. S. (1982) Effect of pregnancy on resistance toListeria monocytogenes and Toxoplasma gondii infections in mice. Infect.Immun. 38, 1164–1171.
9. Matzdorff, A. C., Matthes, K., Kemkes-Matthes, B., Pralle, H. (1997)[Visceral leishmaniasis with an unusually long incubation time]. Dtsch.Med. Wochenschr. 122, 890–894.
10. Meinecke, C. K., Schottelius, J., Oskam, L., Fleischer, B. (1999) Congen-ital transmission of visceral leishmaniasis (Kala Azar) from an asymptom-atic mother to her child. Pediatrics 104, e65.
11. Pagliano, P., Carannante, N., Rossi, M., Gramiccia, M., Gradoni, L.,Faella, F. S., Gaeta, G. B. (2005) Visceral leishmaniasis in pregnancy: acase series and a systematic review of the literature. J. Antimicrob.Chemother. 55, 229–233.
12. Sacks, G., Sargent, I., Redman, C. (1999) An innate view of humanpregnancy. Immunol. Today 20, 114–118.
13. Koumandakis, E., Koumandaki, I., Kaklamani, E., Sparos, L., Aravanti-nos, D., Trichopoulos, D. (1986) Enhanced phagocytosis of mononuclearphagocytes in pregnancy. Br. J. Obstet. Gynaecol. 93, 1150–1154.
14. Luppi, P., Haluszczak, C., Betters, D., Richard, C. A., Trucco, M., DeLoia,J. A. (2002) Monocytes are progressively activated in the circulation ofpregnant women. J. Leukoc. Biol. 72, 874–884.
15. Sacks, G. P., Redman, C. W., Sargent, I. L. (2003) Monocytes are primedto produce the Th1 type cytokine IL-12 in normal human pregnancy: anintracellular flow cytometric analysis of peripheral blood mononuclearcells. Clin. Exp. Immunol. 131, 490–497.
16. Aga, E., Katschinski, D. M., van Zandbergen, G., Laufs, H., Hansen, B.,Muller, K., Solbach, W., Laskay, T. (2002) Inhibition of the spontaneousapoptosis of neutrophil granulocytes by the intracellular parasite Leish-mania major. J. Immunol. 169, 898–905.
17. Laskay, T., van Zandbergen, G., Solbach, W. (2003) Neutrophil granulo-cytes—Trojan horses for Leishmania major and other intracellular mi-crobes? Trends Microbiol. 11, 210–214.
18. Boorman, G. A., Luster, M. I., Dean, J. H., Wilson, R. E. (1980) The effectof adult exposure to diethylstilbestrol in the mouse on macrophage func-tion and numbers. J. Reticuloendothel. Soc. 28, 547–560.
19. Cannon, J. G., St Pierre, B. A. (1997) Gender differences in host defensemechanisms. J. Psychiatr. Res. 31, 99–113.
20. Edwards III, C. K., Schepper, J. M., Yunger, L. M., Kelley, K. W. (1988)Somatotropin and prolactin enhance respiratory burst activity of macro-phages. Ann. N. Y. Acad. Sci. 540, 698–699.
21. Stenger, S., Donhauser, N., Thuring, H., Rollinghoff, M., Bogdan, C.(1996) Reactivation of latent leishmaniasis by inhibition of induciblenitric oxide synthase. J. Exp. Med. 183, 1501–1514.
22. Ali, M., Buhimschi, I., Chwalisz, K., Garfield, R. E. (1997) Changes inexpression of the nitric oxide synthase isoforms in rat uterus and cervixduring pregnancy and parturition. Mol. Hum. Reprod. 3, 995–1003.
23. Conrad, K. P., Joffe, G. M., Kruszyna, H., Kruszyna, R., Rochelle, L. G.,Smith, R. P., Chavez, J. E., Mosher, M. D. (1993) Identification ofincreased nitric oxide biosynthesis during pregnancy in rats. FASEB J. 7,566–571.
24. Stefano, G. B., Prevot, V., Beauvillain, J. C., Fimiani, C., Welters, I.,Cadet, P., Breton, C., Pestel, J., Salzet, M., Bilfinger, T. V. (1999)Estradiol coupling to human monocyte nitric oxide release is dependent onintracellular calcium transients: evidence for an estrogen surface receptor.J. Immunol. 163, 3758–3763.
25. Walsh, L. S., Ollendorff, A., Mershon, J. L. (2003) Estrogen increasesinducible nitric oxide synthase gene expression. Am. J. Obstet. Gynecol.188, 1208–1210.
26. You, H. J., Kim, J. Y., Jeong, H. G. (2003) 17 �-Estradiol increasesinducible nitric oxide synthase expression in macrophages. Biochem.Biophys. Res. Commun. 303, 1129–1134.
27. Chwalisz, K., Winterhager, E., Thienel, T., Garfield, R. E. (1999) Syner-gistic role of nitric oxide and progesterone during the establishment ofpregnancy in the rat. Hum. Reprod. 14, 542–552.
28. Maul, H., Longo, M., Saade, G. R., Garfield, R. E. (2003) Nitric oxide andits role during pregnancy: from ovulation to delivery. Curr. Pharm. Des. 9,359–380.
29. Gomez-Ochoa, P., Gascon, F. M., Lucientes, J., Larraga, V., Castillo, J. A.(2003) Lactating females Syrian hamster (Mesocricetus auratus) showprotection against experimental Leishmania infantum infection. Vet. Para-sitol. 116, 61–64.
30. Satoskar, A., Al-Quassi, H. H., Alexander, J. (1998) Sex-determinedresistance against Leishmania mexicana is associated with the preferentialinduction of a Th1-like response and IFN-� production by female but notmale DBA/2 mice. Immunol. Cell Biol. 76, 159–166.
31. Travi, B. L., Osorio, Y., Melby, P. C., Chandrasekar, B., Arteaga, L.,Saravia, N. G. (2002) Gender is a major determinant of the clinicalevolution and immune response in hamsters infected with Leishmania spp.Infect. Immun. 70, 2288–2296.
32. Osorio, Y., Melby, P. C., Pirmez, C., Chandrasekar, B., Guarin, N., Travi,B. L. (2003) The site of cutaneous infection influences the immunologicalresponse and clinical outcome of hamsters infected with Leishmaniapanamensis. Parasite Immunol. 25, 139–148.
33. Roy, G., Dumas, C., Sereno, D., Wu, Y., Singh, A. K., Tremblay, M. J.,Ouellette, M., Olivier, M., Papadopoulou, B. (2000) Episomal and stableexpression of the luciferase reporter gene for quantifying Leishmania spp.infections in macrophages and in animal models. Mol. Biochem. Parasitol.110, 195–206.
34. Henao, H. H., Osorio, Y., Saravia, N. G., Gomez, A., Travi, B. (2004)[Efficacy and toxicity of pentavalent antimonials (Glucantime and Pento-stam) in an American cutaneous leishmaniasis animal model: luminometryapplication]. Biomedica 24, 393–402.
35. Melby, P. C., Tryon, V. V., Chandrasekar, B., Freeman, G. L. (1998)Cloning of Syrian hamster (Mesocricetus auratus) cytokine cDNAs andanalysis of cytokine mRNA expression in experimental visceral leishman-iasis. Infect. Immun. 66, 2135–2142.
36. Perez, L. E., Chandrasekar, B., Saldarriaga, O. A., Zhao, W., Arteaga,L. T., Travi, B. L., Melby, P. C. (2006) Reduced nitric oxide synthase 2(NOS2) promoter activity in the Syrian hamster renders the animal func-
Osorio et al. Pregnancy and innate immunity in cutaneous leishmaniasis 1421
tionally deficient in NOS2 activity and unable to control an intracellularpathogen. J. Immunol. 176, 5519–5528.
37. Lim, L. C., England, D. M., Glowacki, N. J., DuChateau, B. K., Schell,R. F. (1995) Involvement of CD4� T lymphocytes in induction of severedestructive Lyme arthritis in inbred LSH hamsters. Infect. Immun. 63,4818–4825.
38. Liu, H., Steiner, B. M., Alder, J. D., Baertschy, D. K., Schell, R. F. (1990)Immune T cells sorted by flow cytometry confer protection against infec-tion with Treponema pallidum subsp. pertenue in hamsters. Infect. Immun.58, 1685–1690.
39. Boyum, A. (1974) Separation of blood leucocytes, granulocytes and lym-phocytes. Tissue Antigens 4, 269–274.
40. Lo, F., Kaufman, S. (2001) Effect of 5 �-pregnan-3 �-ol-20-one on nitricoxide biosynthesis and plasma volume in rats. Am. J. Physiol. Regul.Integr. Comp. Physiol. 280, R1902–R1905.
41. Barral, A., Barral-Netto, M., Yong, E. C., Brownell, C. E., Twardzik, D. R.,Reed, S. G. (1993) Transforming growth factor � as a virulence mechanismfor Leishmania braziliensis. Proc. Natl. Acad. Sci. USA 90, 3442–3446.
42. Miller, L., Hunt, J. S. (1996) Sex steroid hormones and macrophagefunction. Life Sci. 59, 1–14.
43. Laufs, H., Muller, K., Fleischer, J., Reiling, N., Jahnke, N., Jensenius,J. C., Solbach, W., Laskay, T. (2002) Intracellular survival of Leishmaniamajor in neutrophil granulocytes after uptake in the absence of heat-labileserum factors. Infect. Immun. 70, 826–835.
44. Ribeiro-Gomes, F. L., Otero, A. C., Gomes, N. A., Moniz-De-Souza, M. C.,Cysne-Finkelstein, L., Arnholdt, A. C., Calich, V. L., Coutinho, S. G.,Lopes, M. F., DosReis, G. A. (2004) Macrophage interactions with neu-trophils regulate Leishmania major infection. J. Immunol. 172, 4454–4462.
45. Van Zandbergen, G., Klinger, M., Mueller, A., Dannenberg, S., Gebert, A.,Solbach, W., Laskay, T. (2004) Cutting edge: neutrophil granulocyte
serves as a vector for Leishmania entry into macrophages. J. Immunol.173, 6521–6525.
46. Melby, P. C., Chandrasekar, B., Zhao, W., Coe, J. E. (2001) The hamsteras a model of human visceral leishmaniasis: progressive disease andimpaired generation of nitric oxide in the face of a prominent Th1-likeresponse. J. Immunol. 166, 1912–1920.
47. Basu, R., Bhaumik, S., Basu, J. M., Naskar, K., De, T., Roy, S. (2005)Kinetoplastid membrane protein-11 DNA vaccination induces completeprotection against both pentavalent antimonial-sensitive and -resistantstrains of Leishmania donovani that correlates with inducible nitric oxidesynthase activity and IL-4 generation: evidence for mixed Th1- andTh2-like responses in visceral leishmaniasis. J. Immunol. 174, 7160–7171.
48. Ramirez-Emiliano, J., Gonzalez-Hernandez, A., Arias-Negrete, S. (2005)Expression of inducible nitric oxide synthase mRNA and nitric oxideproduction during the development of liver abscess in hamster inoculatedwith Entamoeba histolytica. Curr. Microbiol. 50, 299–308.
49. MacRitchie, A. N., Jun, S. S., Chen, Z., German, Z., Yuhanna, I. S.,Sherman, T. S., Shaul, P. W. (1997) Estrogen upregulates endothelialnitric oxide synthase gene expression in fetal pulmonary artery endothe-lium. Circ. Res. 81, 355–362.
50. Karpuzoglu, E., Fenaux, J. B., Phillips, R. A., Lengi, A. J., Elvinger,F., Ansar Ahmed, S. (2006) Estrogen up-regulates inducible nitricoxide synthase, nitric oxide, and cyclooxygenase-2 in splenocytesactivated with T cell stimulants: role of interferon-�. Endocrinology147, 662– 671.
51. Karpuzoglu, E., Phillips, R. A., Gogal Jr., R. M., Ansar Ahmed, S. (2007)IFN-�-inducing transcription factor, T-bet is upregulated by estrogen inmurine splenocytes: role of IL-27 but not IL-12. Mol. Immunol. 44,1808–1814.
1422 Journal of Leukocyte Biology Volume 83, June 2008 http://www.jleukbio.org
