involvement of gonadal steroids and gamma interferon in...

INFECTION AND IMMUNITY, June 2006, p. 3190–3203 Vol. 74, No. 60019-9567/06/$08.00�0 doi:10.1128/IAI.00008-06Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Involvement of Gonadal Steroids and Gamma Interferon in SexDifferences in Response to Blood-Stage Malaria Infection†

Amy Cernetich,‡ Lindsey S. Garver, Anne E. Jedlicka, Pamela W. Klein, Nirbhay Kumar,Alan L. Scott, and Sabra L. Klein*

W. Harry Feinstone Department of Molecular Microbiology and Immunology,The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205

Received 3 January 2006/Returned for modification 11 February 2006/Accepted 4 March 2006

To examine the hormonal and immunological mechanisms that mediate sex differences in susceptibility tomalaria infection, intact and gonadectomized (gdx) C57BL/6 mice were inoculated with Plasmodium chabaudiAS-infected erythrocytes, and the responses to infection were monitored. In addition to reduced mortality,intact females recovered from infection-induced weigh loss and anemia faster than intact males. Expressionmicroarrays and real-time reverse transcription-PCR revealed that gonadally intact females exhibited higherexpression of interleukin-10 (IL-10), IL-15R�, IL-12R�, Gadd45�, gamma interferon (IFN-�), CCL3,CXCL10, CCR5, and several IFN-inducible genes in white blood cells and produced more IFN-� than did intactmales and gdx females, with these differences being most pronounced during peak parasitemia. Intact femalesalso had higher anti-P. chabaudi immunoglobulin G (IgG) and IgG1 responses than either intact males or gdxfemales. To further examine the effector mechanisms mediating sex differences in response to P. chabaudiinfection, responses to infection were compared among male and female wild-type (WT), T-cell-deficient(TCR��/�), B-cell-deficient (�MT), combined T- and B-cell-deficient (RAG1), and IFN-� knockout (IFN-��/�) mice. Males were 3.5 times more likely to die from malaria infection than females, with these differencesbeing most pronounced among TCR��/�, �MT, and RAG1 mice. Male mice also exhibited more severe weightloss, anemia, and hypothermia, and higher peak parasitemia than females during infection, with WT, RAG1,TCR��/�, and �MT mice exhibiting the most pronounced sexual dimorphism. The absence of IFN-� reducedthe sex difference in mortality and was more detrimental to females than males. These data suggest thatdifferential transcription and translation of IFN-�, that is influenced by estrogens, may mediate sex differencesin response to malaria.

Males are more susceptible to many protozoan infectionsthan females and field and laboratory studies link increasedsusceptibility to infection with circulating steroid hormones(17, 18, 39). One genus of protozoan parasites that causes apronounced sexual dimorphism in vertebrate hosts is Plasmo-dium. Among humans, although the incidence of infection isoften similar between the sexes (see references 51 and 52), sexdifferences in the intensity of infection are reported in whichmen have higher parasitemia than women (23, 32, 52). Theobservation that P. falciparum (i.e., a human malaria parasite)density increases at puberty in men, but not in women, suggeststhat circulating sex steroids may influence this outcome (23).

Studies of rodent malarias have confirmed that males aremore likely to die after blood-stage malaria infection than arefemales (2, 3, 54–56). Castration of male mice reduces,whereas exogenous administration of testosterone increases,mortality after infection with P. chabaudi or P. berghei (15, 54).The immunosuppressive effects of testosterone may underlieincreased susceptibility to Plasmodium infections in malescompared to females. Injection of female mice with high doses

of testosterone reduces antibody production, the number ofmajor histocompatibility complex class II cells in the spleen,and the expression of malaria-responsive genes in the liver butdoes not affect cytokine production (2, 22). Receptors for sexsteroids are expressed in various lymphoid tissue cells, as wellas in circulating lymphocytes, macrophages, and dendritic cells(8, 39, 43, 53). The binding of sex steroids to their respectivesteroid receptors directly influences cell signaling pathways,including nuclear factor-�B (NF-�B), resulting in the differen-tial production of cytokines and chemokines by cells of theimmune system (30). Whereas cellular signaling throughNF-�B induces the expression of immune and inflammatorygenes, steroid hormone signaling can antagonize NF-�B-me-diated responses, resulting in tightly regulated communicationbetween the endocrine and immune systems (30). If sex ste-roids influence the sexual dimorphism in immune responses toPlasmodium infection, then removal of the sex steroids viagonadectomy may significantly alter immune and inflammatoryresponses during malaria infection.

Utilization of mice infected with rodent Plasmodium specieshas been instrumental for characterizing the pathogenesis andimmunobiology of blood-stage malaria (46). In mice that areresistant to blood-stage malaria infection, production of inter-leukin-12 (IL-12), tumor necrosis factor (TNF), and gammainterferon (IFN-�) during the acute phase of infection andantibody production during the chronic phase of infection iscritical for recovery from Plasmodium chabaudi infection (46).Studies of human and rodent malarias illustrate that proin-

* Corresponding author. Mailing address: Department of MolecularMicrobiology and Immunology, Johns Hopkins Bloomberg School ofPublic Health, 615 North Wolfe St., Baltimore, MD 21205-2179. Phone:(410) 955-8898. Fax: (410) 955-0105. E-mail: [email protected].

† Supplemental material for this article may be found at http://iai.asm.org/.

‡ Present address: Department of Microbiology and Immunology,Drexel University College of Medicine, Philadelphia, PA 19129.

3190

on May 14, 2018 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

flammatory immune responses are necessary for the develop-ment of protective immunity but must be regulated to preventpathology (24). The timing and shift from Th1 to Th2 re-sponses during the course of Plasmodium infection is mediatedby regulatory responses, including the production of trans-forming growth factor � (TGF-�) and IL-10 (25, 35). A ma-jority of the rodent studies characterizing protective immuneresponses against blood-stage malaria infection have used fe-male mice. Whether the development and timing of protectiveimmune responses during infection differ between males andfemales and are altered by sex steroid hormones has not beenadequately examined.

The primary goal of the present study was to examine sexdifferences in the pathogenesis and immunobiology of P.chabaudi infection in C57BL/6 mice. Gonadally intact femalesare more resistant to infection than males; the mechanismsmediating this sex difference remains elusive. We hypothesizedthat intact females would have reduced parasitemia, anemia,weight loss, and hypothermia during infection compared tomales. Because elevated proinflammatory and regulatory T-cell responses during the acute phase of infection and height-ened antibody responses during the persistent phase of in-fection are associated with protection and recovery frominfection, we hypothesized that these responses would be ele-vated among intact females compared to intact males. Wefurther hypothesized that if sex steroids mediate the sexualdimorphism in infection, then the removal of sex steroids viagonadectomy would reverse the sex difference in response toblood-stage malaria infection. Finally, to provide convergingevidence for the effector responses that mediate sex-baseddifferences in response to malaria, responses to P. chabaudichallenge were compared among male and female T-cell-defi-cient (TCR��/�), B-cell deficient (�MT), T- and B-cell-de-ficient (RAG1), and IFN-� deficient (IFN-��/�) mice. Overall,our data reveal that removal of the ovaries (i.e., the primarysource of estrogens) significantly alters immune responses dur-ing blood-stage malaria infection to a greater extent than re-moval of the testes, suggesting that sex differences in responseto malaria may be estrogen dependent. Our data also illustratethat females rely more heavily on IFN-� than do males toovercome malaria infection, indicating that IFN-� may play animportant role in mediating the sexual dimorphism in resis-tance to blood-stage malaria infection.

MATERIALS AND METHODS

Animals. Adult (�60-day-old) male (n 100; 10/time point/treatment) andfemale (n 100; 10/time point/treatment) C57BL/6 mice were purchased fromthe National Cancer Institute (Bethesda, MD). Adult (�60-day-old) male (n 15/genotype) and female (n 15/genotype) wild-type (WT), T-cell-deficient(TCR��/� mice), B-cell-deficient (�MT mice), combined T- and B-cell-defi-cient (RAG1 mice), and IFN-� knockout (IFN��/� mice) mice, all on a C57BL/6background, were purchased from Jackson Laboratories (Bar Harbor, ME). Allanimals were housed at five mice/cage in a microisolator room and maintainedon a constant light-dark cycle (16 and 8 h). Food and sterile tap water wereavailable ad libitum. The Johns Hopkins Animal Care and Use Committee(protocol MO02H49) and the Johns Hopkins Office of Health, Safety, andEnvironment (P0303310202) approved all procedures described here.

Infection. P. chabaudi chabaudi AS parasites were kindly provided by Mary M.Stevenson (McGill University, Montreal, Quebec, Canada) and were maintainedin donor BALB/c mice by weekly passage from frozen stock cultures. Parasiteswere passaged two to three times prior to use in experimental animals. All

experimental animals received an intraperitoneal (i.p.) inoculation of 106 P.chabaudi chabaudi AS-infected erythrocytes.

Procedures. For gonadectomy, animals were assigned to remain intact or bebilaterally gonadectomized (gdx). Mice were bilaterally gdx under ketamine (80mg/kg [body mass])-xylazine (8 mg/kg [body mass]) anesthesia (Phoenix Phar-maceutical, St. Joseph, MO) and given 2 to 3 weeks to recover from surgery.After infection, animals were killed 0, 3, 7, 14, or 21 days postinoculation (p.i.).At each time point, animals were anesthetized with isoflurane vapors (AbbottLaboratories, Chicago, IL); body mass was measured; and blood was collectedfrom the retro-orbital sinus to assess anemia, parasitemia, and anti-P. chabaudiantibody responses. Spleens were dissected and weighed and white blood cells(WBCs) were isolated and used to measure mRNA expression and proteinproduction. Body mass and blood samples also were collected at 5 and 10 daysp.i. For experiments with mice with selective deficiencies, following i.p. inocula-tion with P. chabaudi-infected erythrocytes, parasitemia, body mass, rectal tem-perature, and anemia were repeatedly monitored 0, 3, 5, 7, 10, 14, and 21 days p.i.The proportion of animals that survived infection also was recorded.

Parasitemia. Thin blood smears were prepared, fixed with methanol, andstained with a 1:10 dilution of Giemsa stain (Sigma, St. Louis, MO) in 1phosphate buffer (3 mM potassium phosphate monobasic, 4.7 mM sodium phos-phate monobasic [pH 7.1]). Parasites were visualized under a 100 oil immer-sion lens, and parasitemia was calculated by counting the number of parasites/total number of erythrocytes in a minimum of three random fields.

Anemia. Blood was collected into heparinized microhematocrit tubes, tubeswere plugged with clay and centrifuged for 15 min at 1,200 rpm, and the redblood cell (RBC) volume was measured relative to the total blood volume.

Body temperature. To monitor body temperature, animals were briefly re-strained in a modified 50-ml conical tube, and rectal temperature was measuredwithin 5 s (Physitemp, Clifton, NJ).

WBC isolation. Spleens were placed in sterile RPMI 1640 medium (MediatechCellgro, Herndon, VA), and WBCs were recovered by pressing the whole spleenthrough a 100-�m-pore-size nylon cell strainer. Separated cells were suspendedin additional RPMI 1640, and erythrocytes were lysed with 10 ml of erythrocytelysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.1mM Na2� EDTA [pH 7.2 to 7.4]). Cells were washed twice, resuspended in 10ml of phosphate-buffered saline (PBS), and WBC counts and viability weredetermined by using a hemacytometer and trypan blue exclusion.

IFN-� ELISA. Viable WBCs were adjusted to 5 106 cells/ml in supple-mented culture medium (RPMI 1640 with 25 mM HEPES, 10% fetal bovineserum, 2 mM L-glutamine, 1% penicillin-streptomycin, 0.2 mM 2-mercaptoeth-anol). Plates were incubated at 37°C with 5% CO2 for 72 h. The cell supernatantwas removed from each well, centrifuged for 5 min at 5,000 rpm, transferred intosterile tubes, and stored at �80°C. IFN-� concentrations were assayed by en-zyme-linked immunosorbent assay (ELISA) by diluting samples to 1:10 and usingthe manufacturer’s protocols for the OptEIA IFN-� kit (BD Pharmingen, SanDiego, CA).

Anti-P. chabaudi ELISA. Lysate of P. chabaudi antigen was used as a captureantigen, and the protein concentration was estimated by using a bicinchoninicacid protein assay (Pierce, Rockford, IL). Microtiter plates were coated over-night at 4°C in carbonate buffer with 5 �g of P. chabaudi antigen/ml for immu-noglobulin G (IgG) or 20 �g of P. chabaudi antigen/ml for IgG1 and IgG2c (45).Plates were blocked by using 5% milk in PBS with 0.05% Tween 20 (PBS-T) forIgG or 2% bovine serum albumin in PBS for IgG1 and IgG2c. Plasma samples,as well as positive and negative control samples, were diluted 1:100 for IgG and1:50 for IgG1 and IgG2c in PBS (sera dilutions were based on optimizationassays). Secondary antibody (horseradish peroxidase [HRP]-conjugated goat anti-mouse IgG [Zymed, South San Francisco, CA], HRP-goat anti-mouse IgG1[Southern Biotechnology Associates, Birmingham, AL], and HRP-goat anti-mouse IgG2c [Southern Biotechnology Associates]) was diluted 1:5,000 in PBSfor IgG and 1:2,000 in 0.5% bovine serum albumin in PBS-T for IgG1 and IgG2c.Tetramethylbenzidine substrate (BD Pharmingen) was added, and the enzyme-substrate reaction was terminated after 30 min by adding 2 N H2SO4. The opticaldensity was measured at 450 nm, and the samples were expressed as a percentageof the positive control run on the same microtiter plate.

Microarrays. Viable WBCs were adjusted to 5 106 cells/ml by dilution withPBS and stored in liquid nitrogen. For RNA extraction, TRIzol LS (Invitrogen,Carlsbad, CA) was added to cells, followed by high-speed disruption in a Fast-Prep 120 Instrument (Q-Biogene, Irvine, CA) at speed 5 for 15 s. Homogenateswere subsequently processed according to the manufacturer’s protocol (Invitro-gen). RNA pellets were resuspended in nuclease-free water. The quantitation ofRNA was performed by using a Beckman DU640 spectrophotometer, and anRNA quality assessment was determined by RNA Nano LabChip analysis on anAgilent Bioanalyzer 2100. Equivalent aliquots (2.5 �g) of RNA from three

VOL. 74, 2006 SEX DIFFERENCE IN MALARIA INFECTION 3191


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

animals/treatment group were pooled, and three separate pools were processedon separate Affymetrix GeneChips (n 3 GeneChips/time point/treatmentgroup) (16). The variability among the three GeneChips, within each treatmentgroup at each time point ranged from 0.83 to 2.25%.

The processing of RNA for GeneChip analysis was in accordance with themethods described in the Affymetrix GeneChip Expression Analysis TechnicalManual, revision three, as previously reported (14). Hybridization cocktails wereprepared as recommended prior to pipetting into the Affymetrix GeneChips(Murine Genome MOE430A). Hybridization and the signal amplification pro-tocol for washing and staining of eukaryotic targets were performed as previouslydescribed (14). The arrays were scanned at an emission wavelength of 570 nm at2.5-mm resolution in the GCS3000 laser scanner (Affymetrix). The intensity ofthe hybridization for each probe pair was computed by GCOS 1.1 software. Formore detailed methods, please refer to the Web site of the Malaria ResearchInstitute Gene Array Core Facility at the Johns Hopkins Bloomberg School ofPublic Health (http://jhmmi.jhsph.edu/). Primary analysis of microarrays con-sisted of a quality assessment of the hybridization for each sample. The ratios ofsignal for probe sets at the 5� and 3� regions of the housekeeping genes werecalculated and monitored as an indication of the transcript quality for eachsample.

Real-time RT-PCR for microarray validation. Custom primer and probe setswere generated for each target gene by using Primer Express 2.0 software(Applied Biosystems, Foster City, CA). Reverse transcription (RT) of 1.5 �g ofRNA from each individual animal sample (n 8 to 10/time point/treatmentgroup) was conducted with oligo(dT)16 primers and according to the manufac-turer’s protocol for the SuperScript II First-Strand Synthesis System for RT-PCR(Invitrogen). cDNA was amplified in a reaction mixture containing 50 �Mconcentrations of the primers, a 250 �M concentration of the probe, and theTaqMan Universal PCR mix according to the manufacturer’s protocol (AppliedBiosystems). All reactions were run in optical 96-well plates using the Prism 7000Sequence Detection System (Applied Biosystems). Serial dilutions of pooledcDNA from randomly selected animals from each treatment group at each timepoint p.i. were used to generate a standard curve, ranging from 0.5 to 100 ng/well,which was run on each plate. Gene expression patterns for each target gene werereported relative to the values from same-treatment uninfected control animals.

Statistical analyses. The proportion of animals that survived infection wascompared among experimental groups by chi-square analyses. Survival curveswere compared between sexes by using Cox Hazard regression analyses andlog-rank analyses. Parasitemia, anemia, body temperature, and body masschanges were analyzed by using mixed analyses of variance (ANOVAs), with onewithin subjects variable (days p.i.) and one between subjects variable (treatmentgroup). Antibody responses and splenomegaly were analyzed by using two-wayANOVAs with two between subjects variable (day p.i. and treatment group).IFN-� concentrations were assessed by using a one-way ANOVA. Significantinteractions were further analyzed by using planned comparisons or the Tukeymethod for pairwise multiple comparisons. The combined contribution of para-sitemia, anemia, weight loss, and hypothermia to mortality was investigated byusing multiple linear regression. Correlations among dependent variables wereassessed by using Pearson product moment correlational analyses. Mean differ-ences were considered statistically significant if P was �0.05.

For microarray analyses, data were preprocessed by using GC-RMA in Gene-Spring 7.2 (13). Gene expression patterns for each gene from infected animalswere normalized to the expression levels from same-treatment uninfected con-trol (i.e., day 0) animals (i.e., per gene normalization) (19). Two-way ANOVAs(the variables being the day p.i. and the treatment group) were used to comparegene expression patterns between intact males and females and between gdx andintact animals of the same sex, and mean differences were considered statisticallysignificant if P was �0.05. Because thousands of statistical tests were performedon the data set, false rejection of the null hypothesis is likely to occur. Therefore,we calculated the false discovery rate (47) for our data set and determined thatfor the genes that had a statistically significant difference, the false discoveryrates for each comparison were as follows: intact males versus intact females 0.15; intact males versus gdx males 0.26; and intact females versus gdx females 0.09. All gene expression data from the Affymetrix output are located on theNCBI Gene Expression Omnibus (GEO accession GSE4324; www.ncbi.nlm.nih.gov/geo/).

RESULTS

Intact males are more likely to die from infection and re-cover more slowly from infection-induced weight loss and ane-mia than intact females. To determine whether responses to

blood-stage malaria differed between the sexes and were af-fected by the presence or absence of gonadal steroids, intactand gdx C57BL/6 mice were inoculated with P. chabaudi AS-infected erythrocytes, and body mass, anemia, parasitemia,antibody responses, IFN-� concentrations, and expression lev-els of immune-related genes were monitored. Significantlymore intact C57BL/6 males (7 of 25) died from P. chabaudiinfection than gdx males (0 of 20), intact females (1 of 24), andgdx females (1 of 21) (P 0.005; Fig. 1A). Death from P.chabaudi infection occurred between days 8 and 10 p.i. Levelsof parasitemia peaked 7 days after inoculation with P. chabaudifor all animals (P � 0.0001; Fig. 1B). The peak parasitemia wasmarginally higher for gdx males than for intact males, intactfemales, or gdx females (P 0.07; Fig. 1B).

Body mass and RBC turnover are affected by sex steroids(21, 50); therefore, we examined these responses from infectedanimals relative to the responses from uninfected same sex andtreatment controls. After inoculation with P. chabaudi, bodymass decreased significantly through day 10 p.i. and returnedto baseline by day 21 p.i. for all animals (P � 0.0001; Fig. 1C).The body mass of intact females, however, returned to baselinefaster than for intact males and gdx females, as noted by therapid weight gain among intact females 14 and 21 days p.i.(P � 0.05; Fig. 1C). After inoculation with P. chabaudi, the per-centage of RBCs was lower for all animals at 10 days p.i.compared to values at other time points p.i. (P � 0.0001; Fig.1D). There was a trend for intact females to recover fromanemia faster than intact males and gdx females, as indicatedby the rapid replacement of RBC in intact females by 14 daysp.i. (P 0.06; Fig. 1D). Over the course of infection, the spleenmass increased, whereas the WBC counts decreased, for allanimals by 14 and 21 days p.i. (P � 0.001 in each case; data notshown). There were no sex differences or effects of gonadec-tomy on splenomegaly or the numbers of WBCs during P.chabaudi infection (P � 0.05).

During the acute phase of infection, the expression of IFN-�-associated and regulatory genes differs between intact malesand females and is altered by gonadectomy. Expression mi-croarrays and real-time RT-PCR were used to measurechanges in gene expression in WBCs 0, 3, 7, 14, and 21 days p.i.with P. chabaudi. Prior to infection (day 0), of the 22,690 genesarrayed on the mouse MOE430A GeneChip (Affymetrix),492 genes showed differential expression levels between in-tact males and intact females, 45 genes were differentiallyexpressed between intact males and gdx males, and 3 genesdiffered between intact females and gdx females. Becausebaseline differences in gene expression were observed, geneexpression patterns for each gene from infected animalswere normalized to the expression levels from same-treat-ment uninfected control (i.e., day 0) animals (i.e., per genenormalization) (19), which enabled sex or treatment differ-ences in the expression levels of genes 3 to 14 days p.i. to beattributed to infection.

During infection (days 3 to 14 p.i.), 5,182 genes showedsignificant differences in the relative expression intensity be-tween the sexes, 4,360 genes differed between intact males andgdx males, and 7,013 genes differed between intact females andgdx females (P � 0.05 in each case). Using GO BiologicalProcesses descriptions, we identified 74 unique genes with as-sociated immune function that had expression levels that dif-

3192 CERNETICH ET AL. INFECT. IMMUN.


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

fered significantly between intact males and intact femalesduring infection (P � 0.05; Fig. 2A and Table S1 in the sup-plemental material); 53 unique immune-related genes that dif-fered significantly between intact males and gdx males duringinfection (P � 0.05; Fig. 2B and Table S2 in the supplementalmaterial), and 60 unique immune-related genes that differedsignificantly between intact females and gdx females during P.chabaudi infection (P � 0.05; Fig. 2C and Table S3 in thesupplemental material). Differential expression of genes dur-ing P. chabaudi infection was observed 3, 7, and 14 days p.i.(Tables S1 to S3 in the supplemental material); differences ingene expression levels among the groups of mice, however, weremost pronounced during peak parasitemia (i.e., day 7 p.i.; Fig. 2).

Among intact mice, males and females exhibited sexuallydimorphic expression of several genes associated with innateimmunity, antigen presentation, cell adhesion, cytokines, ironmetabolism, apoptosis, cellular proliferation, complement, andantibody production during P. chabaudi infection (P � 0.05;Fig. 2A and Table S1 in the supplemental material). Of specificinterest, genes involved in the production of IFN-� and thedevelopment of Th1 immunity were consistently upregulatedamong intact female mice during peak parasitemia. For exam-ple, CCL3 (i.e., MIP-1 ), CXCL10 (i.e., IP-10), CCR5, IFN-�,growth arrest- and DNA-damage-inducible 45� (Gadd45�),IL-12R�2, and IL-15R , as well as IFN /�/�-inducible genes(e.g., IFN-�-inducible protein 16 [Ifi16] and IFN-induced pro-tein with tetratricopeptide repeats 2 [Ifit2]) had significantly

higher expression levels in intact females than intact malesduring peak parasitemia (Fig. 2A and Table S1 in the supple-mental material). In particular, the expression of IFN-� wassixfold higher in intact females than in intact males duringpeak parasitemia (Fig. 3C). In addition, the expression of reg-ulatory T-cell genes, including IL-10 and TNFR superfamilymember 4 (Tnfrsf4; OX-40), was twofold higher among intactfemale than intact male mice during peak parasitemia (Fig. 4Aand B). IFN-� inhibits iron metabolism in WBCs (4) and sev-eral genes associated with iron regulation, including lactotrans-ferrin (Ltf) and transferrin receptor 2 (TrfR2), were signifi-cantly downregulated among intact females compared to intactmales (P � 0.05; Fig. 5A and B.

During infection, gonadectomy of male and female micesignificantly altered the expression of immune-related genesrelative to their intact counterparts. Gonadectomy of male andfemale mice significantly altered the expression of genes asso-ciated with innate immunity, chemokines, cytokines, antigenpresention, regulatory responses, cellular proliferation, apop-tosis, and signal transduction compared to their intact coun-terparts (P � 0.05; Fig. 2B and C and Tables S2 and S3 in thesupplemental material). Gonadectomy of male mice increasedthe expression of IL-15R , Gadd45�, IFN-induced trans-membrane protein 3-like (Ifitm3l), IFN-�-inducible Puma-g(Gpr109b), Myd88, CCL3, IFN-related developmental regula-tor 1 (Ifrd1), and TGF-� induced (Tgfbi) compared to intactmales (Fig. 2B and Table S2 in the supplemental material).

FIG. 1. (A) Cumulative survival; (B) percent (mean � the standard error of the mean [SEM]) parasitemia (parasitized RBC/total RBC);(C) average (mean � the SEM) change in body mass (grams) from baseline (day 0); (D) average (mean � the SEM) change in the percentageof RBC from baseline (day 0) for intact males, gdx males, intact females, and gdx females 3, 5, 7, 10, 14, and 21 days p.i. with P. chabaudi parasites.❋, intact females versus intact males; †, intact females versus gdx females, P � 0.05 (as determined by �2 [A] and mixed ANOVA [B to D] analyses).



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

Gonadectomy of female mice significantly suppressed the ex-pression of several genes associated with Th1 immunity andregulatory T-cell responses, including Ifrd1, TGF-�-inducibleearly growth response 1 (Tieg1) CCR5, IFN-�, Gadd45�, IL-12R�2, and IL-15R , during infection (Fig. 2C and Table S3 inthe supplemental material).

Because of the potential problem of false positives associ-ated with microarray analyses, several genes of interest werevalidated by using real-time RT-PCR. Real-time RT-PCR alsowas used to further examine gene expression profiles at 21 daysp.i. The expression patterns of IL-12R�2, IFN-�, Gadd45�,IL-10, Tnfrsf4, Ltf, TrfR, and -actin in WBCs from individualanimals were examined, and the expression levels of each genefrom infected animals were analyzed relative to the expressionlevels of each gene from uninfected same-treatment controlanimals. The expression of the housekeeping gene -actin wassimilar among intact and gdx males and females days 0 to21 p.i. (P � 0.05). Consistent with our microarray results, theexpression of genes associated with the synthesis of IFN-�,including IL-12R�, Gadd45�, and IFN-�, was higher amongintact females than intact males, gdx males, and gdx females7 days p.i. (P � 0.01 in each case; Fig. 3). Although microar-ray analyses revealed that gdx males had a higher expressionof Gadd45� than intact males at 3 and 7 days p.i. (Fig. 3B),this observation was not replicated with real-time RT-PCR(Fig. 3E).

During peak parasitemia (i.e., day 7 p.i.), WBCs isolatedfrom intact females produced more IFN-� than did WBCsisolated from intact males and gdx females (P � 0.01; Fig. 3F).Animals with the highest IFN-� concentrations tended to have

the lowest parasitemia (r �0.29, P 0.06) and the highestexpression of IFN-� (r 0.44, P � 0.005), Gadd45� (r 0.69,P � 0.0001), IL-12R� (r 0.51, P � 0.001), and IL-10 (r 0.56, P � 0.0001) at day 7 p.i.

Microarray and real-time RT-PCR revealed that intact fe-males had higher expression of regulatory genes, includingIL-10 and Tnfrsf4, than intact males during peak parasitemia(P � 0.001 in each case; Fig. 4); real-time RT-PCR resultsfurther indicated that gonadectomy of females significantlysuppressed the expression of IL-10 and Tnfrsf4 and gonadec-tomy of males enhanced the expression of IL-10 at day 7 p.i.(Fig. 4C and D). Microarray and real-time RT-PCR analysesconsistently illustrated that the expression of genes associatedwith iron homeostasis, including Ltf and TrfR, was higheramong intact males than among intact females 14 days p.i. andthat gonadectomy of male mice significantly reduced the ex-pression of the TrfR (P � 0.001 in each case; Fig. 5).

During the recovery phase of infection, intact females havehigher antibody responses than either intact males or gdxfemales. To eliminate the erythrocytic stage of Plasmodiumparasites, both humoral and cell-mediated effector mecha-nisms are required. Anti-P. chabaudi IgG increased over timefor all animals and peaked at 14 to 21 days p.i. (P � 0.001; Fig.6A). Anti-P. chabaudi IgG antibody responses, however, werehigher among intact females than among intact males at 14 to21 days p.i; gonadectomy of female mice reversed this sexdifference by 21 days p.i. (P � 0.01; Fig. 6A).

To assess whether different subsets of helper T cells wereactivated by males and females in response to infection, sub-classes of IgG were examined. Anti-P. chabaudi IgG1 re-

FIG. 2. Differential expression of immune-related genes in WBCs collected during peak parasitemia (i.e., day 7 p.i.) from intact male and intactfemale mice (A), intact male and gdx male mice (B), and intact female and gdx female mice (C). These unique genes were selected from the listsof genes with expression levels that were significantly different between the comparison groups using a two-way ANOVA (P � 0.05; �2-folddifference in the relative expression intensity between the sexes or treatment groups). For each gene, expression data from infected animals werenormalized to the expression levels from same-sex uninfected control animals (i.e., day 0). The hierarchical gene tree illustrates the expressionpatterns of genes using standard correlation analyses, with a separation ratio of 0.5 and minimum distance of 0.001. Genes that are significantlyupregulated are indicated by gradations of red, genes that are significantly downregulated are illustrated by gradations of blue, and genes that donot change after infection are indicated by gradations of yellow. See the text and supplemental Tables S1 to S3 for abbreviations.



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

sponses increased over the course of infection and were high-est at 14 to 21 days p.i. for all animals (P � 0.001; Fig. 6B).Anti-P. chabaudi IgG1 responses were significantly higheramong intact females than intact males at 14 days p.i. (P �0.01; Fig. 6B). C57BL/6 mice do not possess the gene for IgG2abut express a novel isotype, IgG2c, that is indicative of a Th1-

mediated response (28). Anti-P. chabaudi IgG2c responsespeaked 14 to 21 days p.i. for all animals (P � 0.001; Fig. 6C)but did not differ significantly among the experimental groupsof mice (P � 0.05; Fig. 6C).

Utilization of immunodeficient mice reveals that innate im-munity may mediate sexually dimorphic responses to P.

FIG. 3. The expression and translation of genes associated with the synthesis of IFN-� is upregulated in intact females compared to intactmales, and removal of the ovaries in females downregulates the expression of these genes during peak parasitemia (i.e., day 7 p.i.; ❋, intact femalesversus intact males; †, intact females versus gdx females, P � 0.05 [two-way ANOVA]). Gene expression data from infected animals werenormalized to the expression levels from same-treatment uninfected control animals (i.e., day 0). Microarray analyses (A to C) and real-timeRT-PCR analyses (D to F) reveal similar patterns of gene expression. WBCs collected from intact female mice spontaneously produce more IFN-�than do WBCs from intact male and gdx female mice during peak parasitemia (F; ❋, intact females versus intact males; †, intact females versusgdx females, P � 0.05 [one-way ANOVA]).



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

chabaudi infection. To determine the roles of innate and ac-quired immune responses, as well as the specific effects ofIFN-�, as mediators of sex differences in P. chabaudi infection,we inoculated adult WT, TCR��/�, �MT, RAG1, and IFN-��/� mice with P. chabaudi parasites and monitored sex dif-ferences in parasitemia, body mass, rectal temperature, ane-mia, and mortality. Peak parasitemia occurred for all mice at 7days p.i. and was followed by a period of reduced parasitemiain WT, RAG1, and �MT mice (Fig. 7). In contrast, amongTCR��/� and IFN��/� mice, peak parasitemia on day 7 p.i.was followed by parasite recrudescence on day 14 p.i. (Fig. 7).Peak parasitemia was significantly higher among male WT,�MT, and IFN-��/� mice compared to their female counter-parts (P � 0.05 in each case; Fig. 7). Among TCR��/� mice,although peak parasitemia was similar between males and fe-males, males had a higher proportion of parasitized erythro-cytes at 10 days p.i. (because only one TCR��/� male was stillalive days 14 to 21 p.i, statistical comparisons could not bemade at these time points) (P � 0.05; Fig. 7). Peak parasitemiawas similarly high for both male and female RAG1 mice (P �0.05; Fig. 7).

A Cox Hazard regression analysis across all strains of micerevealed that males were 3.5 times (95% confidence interval of2.13 to 5.71) more likely to die from blood-stage malaria in-

fection than were females. Based on log-rank analyses, survivalwas significantly prolonged for female compared to maleRAG1, TCR��/�, and �MT mice (P � 0.001 in each case;Fig. 7). As a result, the average day of death was significantlyearlier for male than female RAG1 (P � 0.01 [males, 8 daysp.i.; females, 14 days p.i.]), TCR��/� (P � 0.001 [males, 10days p.i.; females, 21 days p.i.]), and �MT (P � 0.01 [males, 8days p.i.; females, 13 days p.i.]) mice based on Wilcoxon tests.A similar percentage of male (47%) and female (53%)IFN��/� mice survived infection (P � 0.05), although maleIFN-��/� mice began dying significantly sooner after inocula-tion with P. chabaudi than did females (P � 0.05; Fig. 7).Among male mice, deletion of T cells, B cells, or both lym-phocyte populations significantly increased the likelihood ofdying from blood-stage malaria infection compared to WTmice (P � 0.001 in each case). Among female mice, the ab-sence of IFN-�, B cells, or both T and B cells significantlyincreased mortality from P. chabaudi infection compared toWT females (P � 0.05 in each case).

Consistent with data presented in Fig. 1, among WT mice,females lost less body mass, had less severe anemia, and hadless severe hypothermia during infection and generally re-turned to baseline faster than males (P � 0.05 in each case;Fig. 8). Among RAG1 and TCR��/� mice, males lost more

FIG. 4. Microarray (A to B) and real-time RT-PCR (C to D) analyses illustrate that IL-10 and Tnfrsf4 expression is higher in intact femalesthan intact males and is reversed by gonadectomy during peak parasitemia (i.e., day 7 p.i.; ❋, intact females versus intact males; †, intact femalesversus gdx females; ‡, intact males versus gdx males, P � 0.05 [two-way ANOVA]). Gene expression data from infected animals were normalizedto the expression levels from same-treatment uninfected control animals (i.e., day 0).



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

weight than females by 3 to 7 days p.i. (P � 0.05; Fig. 8).Female RAG1, �MT, and TCR��/� mice consistently devel-oped less severe hypothermia than their male counterparts(P � 0.01 in each case; Fig. 8). Male and female RAG1, �MT,and TCR��/� mice exhibited a similar decrease in the per-centage of RBCs through day 7 p.i. (P � 0.05 in each case; Fig.8). Among IFN-��/� mice, females exhibited an initial de-crease in body mass, the percentage of RBCs, and body tem-perature that was followed by an early return to baseline by day10 p.i. and a subsequent relapse 14 days p.i., resulting in apattern that was significantly different from that of the WTfemale mice (P � 0.01 in each case). Conversely, male IFN-��/� mice demonstrated a more consistent decline in bodymass, percentage of RBC, and body temperature through day10 p.i., followed by a return to baseline by day 14 p.i., whichwas not significantly different from WT males (P � 0.05 in eachcase). The dimorphic response of IFN-��/� mice to infectionresulted in IFN-��/� males having more severe weight loss,anemia, and hypothermia than IFN-��/� females at 10 daysp.i. and IFN-��/� females exhibiting increased morbidity 14days p.i. (P � 0.001 in each case; Fig. 8).

Multiple linear regression analyses revealed that at 7 daysp.i. parasitemia, but not anemia, weight loss, or hypothermia,was a significant predictor of death from P. chabaudi in males

(P � 0.001) and tended to predict death in females (P 0.06).By day 10 p.i., death from P. chabaudi was predicted by hypo-thermia, but not parasitemia, anemia, or weight loss, in bothmales (P � 0.001) and females (P � 0.001), regardless of theirimmunodeficiency.

DISCUSSION

In the present series of studies, gonadally intact WT maleswere more likely than intact WT females to die after P.chabaudi infection, exhibit slower recovery from infection-as-sociated weight loss, hypothermia, and anemia, have reducedIFN-�-associated gene expression and IFN-� production dur-ing peak parasitemia, and produce less antibody during therecovery phase of infection. Gonadectomy of male and femaleWT mice altered these sex-associated differences, suggestingthat sex steroid hormone, in particular androgens and estro-gens, may modulate immune responses to infection.

Infection of C57BL/6 mice with P. chabaudi causes transientanemia, weight loss, and hypothermia. These sequelae, com-bined with elevated parasitemia, are associated with increasedmortality from blood-stage malaria infection (42). In thepresent study, elevated parasitemia at 7 days p.i. and severehypothermia at 10 days p.i. were significant predictors of death

FIG. 5. Microarray (A and B) and real-time RT-PCR (C and D) analyses illustrate that the expression of Ltf and TrfR is significantly higherin intact males than in intact females, and the removal of the testes in males significantly reduces the expression of TrfR mRNA during the recoveryphase of infection (i.e., day 14 p.i.; ❋, intact females versus intact males; †, intact females versus gdx females; ‡, intact males versus gdx males, P �0.05 [two-way ANOVA]). Gene expression data from infected animals were normalized to the expression levels from same-treatment uninfectedcontrol animals (i.e., day 0).



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

from P. chabaudi in both male and female mice. Susceptibilityto P. chabaudi also is associated with low inflammatory re-sponses during the acute phase of infection (46). We observedthat individuals with the highest concentrations of IFN-�tended to have the lowest levels of parasitemia, which maycontribute to the increased likelihood of surviving infectionamong females compared to males.

Characterization of the immunological causes of sex differ-ences in Plasmodium infection using immunodeficient micerevealed that sex differences in parasitemia, morbidity (as mea-sured by changes body mass, RBC loss, and development ofhypothermia), and mortality were still apparent in the absenceof T cells, B cells, or both cell populations. Resistance to

blood-stage malaria was compromised in immunodeficientmale and female mice relative to their WT counterparts, sinceboth male and female mice deficient in T cells, B cells, or bothcell populations experienced chronic parasitemia and in-creased mortality compared to WT mice. Thus, acquired im-munity plays a critical role in mediating the clearance of par-asites and recovery from infection. These immunodeficiencies,however, were consistently more detrimental to males thanto females because the morbidity and mortality rates, as wellas the levels of parasitemia, were still higher in RAG1,TCR��/�, and �MT male mice than in female mice. Ourdata suggest an important role of innate immunity, includingthe activity of macrophages, dendritic cells, and NK cells (all ofwhich are functional in RAG1, TCR��/�, and �MT mice),in mediating the sexual dimorphism in susceptibility to P.chabaudi.

To further assess immunological differences in response tomalaria infection, microarrays and real-time RT-PCR wereused to map the expression levels of immune-related genes inWBCs over the course of P. chabaudi infection. Previous stud-ies illustrate that the administration of pharmacological dosesof testosterone suppresses the expression of immune-relatedgenes, including Gadd45� and CXCL10, in the livers, but hasno effect on gene expression in the spleens, of female miceinfected with P. chabaudi and tested at day 8 p.i. only (22).Female mice injected with high doses of testosterone alsoexhibit reduced antibody production and expression of majorhistocompatibility complex class II on spleen cells but do notshow altered cytokine production in the spleen during theacute phase of infection compared to intact female mice in-jected with vehicle (2). Whether immunological parametersdiffer between males and females during infection has not beensystematically examined over the course of infection prior tothe present study. We demonstrate that females respond to P.chabaudi infection with a heightened IFN-�-mediated re-sponse during the acute phase of infection, an elevated anti-body response during the chronic phase of infection, and re-duced manifestation of disease compared to males. We alsodemonstrate that gonadectomy of females reverses the female-typic immune response to infection, suggesting that estradiolmay play a more central role than previously assumed. As aresult, our data demonstrate that, in addition to the role playedby testosterone, estrogens may play a pivotal role in immunemodulation of malaria infection.

The timing of Th1 and Th2 responses is critical for protec-tion against many Plasmodium parasites (26, 46). ElevatedTh1-mediated responses during the acute phase of infectionare required to reduce peak parasitemia, and activation ofTh2-mediated responses during the chronic phase of infectionis required to clear parasites (46). Macrophage and dendriticcell-derived IL-12 is important for the development of Th1phenotypic responses, such as the production of IFN-�. Micethat are resistant to blood-stage Plasmodium infection producemore splenic IL-12 and IFN-� prior to and during peak para-sitemia than do susceptible mice during P. chabaudi infection(41, 48, 49). In the present study, intact females exhibitedhigher expression of IFN-�-associated genes, including IL-12R�2 and IFN-�, and produced more IFN-� than did intactmales during peak parasitemia. Genes involved in the synthesisof IFN-�, including Gadd45�, which induces phosphorylation

FIG. 6. Average (mean � the SEM) anti-P. chabaudi IgG (A),IgG1 (B), and IgG2c (C) responses from intact males, gdx males, intactfemales, and gdx females at 0, 3, 7, 14, and 21 days p.i. with P. chabaudiparasites. ❋, intact females versus intact males; †, intact females versusgdx females, P � 0.05 (two-way ANOVA).



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

FIG. 7. Cumulative survival and proportion of parasitized red blood cells in male and female WT, RAG1, TCR��/�, �MT, and IFN��/� miceafter i.p. inoculation with 106 P. chabaudi-infected RBCs. Blood samples were collected 3, 5, 7, 10, 14, and 21 days p.i. Parasitemia data (parasitizedRBC/total RBC) are presented as means � the SEM of the surviving animals at each time point p.i. (starting n 15/sex/genotype). ❋, P � 0.05(mixed ANOVA).



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

of STAT4 by the p38 pathway (33), showed a similar pattern ofexpression. IL-15 promotes Th-1-dependent immunity againstP. chabaudi infection (12), and the expression of IL-15R was higher on WBCs from intact females than from intactmales and was reversed by gonadectomy of both sexes duringpeak parasitemia. Several chemokines, including CCL3 andCXCL10, that preferentially attract Th1 cells (40), were up-regulated in the WBCs from intact females. The chemokine

receptor CCR5, which binds CCL3, is expressed at high levelson Th1 cells (27), and is regulated by estrogens (31), also wasupregulated on WBCs from intact females. The removal ofestrogens via gonadectomy significantly reduced the expressionof these Th1-related genes and IFN-� production in a mannersimilar to the reported effects of estradiol on IFN-� responsesagainst Leishmania mexicana (44).

If IFN-� is the primary mediator of sex differences in re-

FIG. 8. Average (mean � the SEM) change in the percent body mass from baseline (day 0); average (mean � the SEM) change in thepercentage of RBC from baseline (day 0); and average (mean � the SEM) rectal temperature (°C) for male and female WT, RAG1, TCR��/�,�MT, and IFN-��/� mice 3, 5, 7, 10, 14, and 21 days p.i. with P. chabaudi parasites. ❋, P � 0.05 (mixed ANOVA).



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

sponse to P. chabaudi, then elimination of IFN-� should re-duce the sexual dimorphism. Although IFN-��/� males hadhigher peak parasitemia than did conspecific females, similarproportions of male and female IFN-��/� mice died afterinfection, providing further evidence that sex differences dur-ing blood-stage malaria infection may be influenced by theproduction of IFN-�. The removal of IFN-� was consistentlymore detrimental to females than to males, as illustrated by theincreased mortality rates and relapse in body mass loss, RBCloss, and hypothermia in female IFN-��/� compared to femaleWT mice. It should be noted, however, that our observation ofa similar rate of survival among male and female IFN-��/�

mice after P. chabaudi AS inoculation is in contrast to previousdata illustrating that more male than female IFN-��/� mice,on a C57BL/6 background, die during P. chabaudi AS infection(48). Although important, IFN-� is likely not the sole mediatorof sex differences in response to blood-stage malaria infection.

After peak parasitemia, Th1 responses begin to decline andTh2 phenotypic responses increase in mice that are resistant toPlasmodium infection (46). Intact females had significantlyhigher anti-P. chabaudi IgG1 responses than males 14 daysafter infection, suggesting that Th2 responses may be elevatedamong intact females during the recovery phase of infection.Among intact females, elevated IFN-� during the acute phaseof infection may induce increased antibody production duringlater phases of malaria infection (49). Overall, intact femalesdeveloped higher antibody responses than intact males, andremoval of the ovaries and, hence, the primary source of es-trogens, significantly suppressed antibody production. Conse-quently, estrogens potentiate (10) and the administration oftestosterone to female mice reduces (2) antibody productionby B cells. Although intact females produced higher antibodyresponses against P. chabaudi, immunoglobulin gene tran-scripts were elevated in WBCs from intact males, suggestingthat there are posttranscriptional differences between thesexes. Although antibody production was suppressed in intactWT males, the removal of B cells in �MT mice did not abolishthe sexual dimorphism in response to P. chabaudi infection,suggesting that B cells may not be critical mediators of sexdifferences in malaria infection.

Regulatory responses, including the production of TGF-�and IL-10, mediate the timing and shift from Th1 to Th2responses and are critical for mitigating pathology caused byprolonged elevation of inflammatory responses during thecourse of malaria infection (25, 35). Although sex differencesin TGF-� expression were not observed, IL-10 expression washigher in WBCs from intact females than from intact males,and gonadectomy of females reduced, whereas gonadectomyof males increased, the transcription of IL-10. Whether thedifferential expression of IL-10 is mediated by macrophages, Tcells, or both requires additional investigation. The expressionof several genes associated with activated CD4� CD25� T cells(29), including Tnfrsf4, Ltb, CCL3, and Gadd45�, was higheramong intact females than among intact males and was re-duced by gonadectomy of female mice in the present study.Estrogens can augment the expansion of CD4� CD25� T cellsin mice (38). Previous data illustrate that administration ofhigh doses of testosterone to intact females has no effect onproduction of IFN-� or IL-10 (2); thus, sex differences in cy-tokine and possibly chemokine production during malaria in-

fection are likely mediated by estrogens and not androgens, aspreviously hypothesized.

In addition to being a key player in Th1 immunity againstmalaria, IFN-� plays a critical role in iron metabolism inWBCs. IFN-� reduces iron uptake by macrophages both indi-rectly through nitric oxide and iron-regulatory proteins anddirectly by causing downregulation of iron receptors, such asthe TrfR (4). In the present study, the transcription of TrfRwas downregulated among intact females and gdx males com-pared to intact males. Elevated IFN-� production among intactfemales may be one mechanism mediating reduced expressionof the TrfR. Whether IFN-� alters TrfR expression directly orthrough NO-mediated degradation of iron regulatory proteinsrequires additional investigation. In the present study, induc-ible NO synthase expression did not differ between the sexes,suggesting that the direct effects of IFN-� on TrfR expressionmay be involved. Although iron supplementation is beneficialfor resolving anemia, iron chelation, which may be regulated byIFN-�, effectively reduces the incidence and severity of P.falciparum infection in children (9, 36). In mice, iron-supple-mented diets increase parasitemia and mortality following P.chabaudi infection to a greater extent among males than fe-males (11), suggesting that dimorphic iron regulation may con-tribute to differential responses to infection.

Although sex differences in response to malaria infection arereported among both adults and children, little is known aboutthe mechanisms mediating these differences or whether thesesex differences will affect responses to drug treatments or vac-cines. Men and women differ in disease manifestations afterinfection; men are more likely to develop lymphomas, whereaswomen are more likely to develop anemia, after infection (5, 6,34). In general, most studies of malaria in human populationsdo not distinguish between the responses of males and femalesand, thus, the prevalence of sex differences may be underre-ported (1). A few studies do, however, clearly illustrate thatmen are more susceptible than women. For example, severalstudies indicate that men tend to have higher parasitemia thanwomen (23, 32, 52). In a recent prospective study of importedmalaria cases in France, men reported more severe symptomsof malaria infection (e.g., chills, fever, and low platelet counts)than did women (7). Among Ghanaian school children, al-though the prevalence of P. falciparum infection does not differbetween the sexes, the parasite density is significantly higherfor boys than girls around puberty (i.e., from ages 8 to 16),suggesting that circulating sex steroids may influence this out-come (23). Although significantly more men die from infec-tious diseases than women (37), sex differences in mortalityfrom Plasmodium infection are not consistently reported.There are contradictory findings from a study in India, whereinmortality rates were reportedly higher in women than menafter infection with P. falciparum (20).

Our studies with a rodent malaria model indicate that malesand females respond differently to malaria infection. Reducedrates of parasitemia and faster recovery from hypothermialikely contribute to reduced death rates among females com-pared to males. The immunological mechanisms that underliethe dimorphism in morbidity and mortality likely involve theheightened IFN-� and regulatory T-cell response observed infemale mice. These data make a compelling case for the re-evaluation of previous epidemiological studies as well as de-



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

velopment of prospective studies that are designed to system-atically test the hypothesis that males and females differ intheir responses to malaria infection. Although sex differencesin mortality and parasitemia following Plasmodium infectionare reported among rodents, the immunological mechanismsmediating these differences have not been adequately exam-ined. The data from the present study provide candidate im-munological pathways that differ between the sexes and thatare significantly altered by the absence of estrogens duringblood-stage malaria infection.

ACKNOWLEDGMENTS

We thank Mary Stevenson and Mifong Tam for providing the initialstock of P. chabaudi parasites and for assistance with assay develop-ment. We also thank Marie Diener-West and Gregory Glass for sta-tistical advice and Judith Easterbrook, Autumn Girouard, MicheleHannah, Peter Klein, and Brandon Zonker for technical assistance.

Financial support for this study was provided by The Johns HopkinsMalaria Research Institute.

REFERENCES

1. Allotey, P., and M. Gyapong. 2005. The gender agenda in the control oftropical diseases: a review of current evidence. World Health OrganizationSpecial Programme for Research, Geneva, Switzerland.

2. Benten, W. P., P. Ulrich, W. N. Kuhn-Velten, H. W. Vohr, and F. Wunderlich.1997. Testosterone-induced susceptibility to Plasmodium chabaudi malaria:persistence after withdrawal of testosterone. J. Endocrinol. 153:275–281.

3. Benten, W. P., F. Wunderlich, and H. Mossmann. 1992. Testosterone-in-duced suppression of self-healing Plasmodium chabaudi malaria: an effectnot mediated by androgen receptors? J. Endocrinol. 135:407–413.

4. Bouton, C., and J. C. Drapier. 2003. Iron regulatory proteins as NO signaltransducers. Sci. STKE 2003:pe17.

5. Brabin, B. J. 1990. An analysis of malaria parasite rates in infants: 40 yearsafter MacDonald. Bureau Hyg. Trop. Dise. 87:R1–R21.

6. Brabin, B. J., L. Brabin, G. Crane, K. P. Forsyth, M. P. Alpers, and H. J. vander Kaay. 1989. Two populations of women with high and low spleen ratesliving in the same area of Madang, Papua New Guinea, demonstrate differ-ent immune responses to malaria. Trans. R. Soc. Trop. Med. Hyg. 83:577–583.

7. Casalino, E., J. Le Bras, F. Chaussin, A. Fichelle, and E. Bouvet. 2002.Predictive factors of malaria in travelers to areas where malaria is endemic.Arch. Intern. Med. 162:1625–1630.

8. Cutolo, M., S. Accardo, B. Villaggio, A. Barone, A. Sulli, D. A. Coviello, C.Carabbio, L. Felli, D. Miceli, R. Farruggio, G. Carruba, and L. Castagnetta.1996. Androgen and estrogen receptors are present in primary cultures ofhuman synovial macrophages. J. Clin. Endocrinol. Metab. 81:820–827.

9. Fritsche, G., C. Larcher, H. Schennach, and G. Weiss. 2001. Regulatoryinteractions between iron and nitric oxide metabolism for immune defenseagainst Plasmodium falciparum infection. J. Infect. Dis. 183:1388–1394.

10. Grimaldi, C. M., L. Hill, X. Xu, E. Peeva, and B. Diamond. 2005. Hormonalmodulation of B-cell development and repertoire selection. Mol. Immunol.42:811–820.

11. Harvey, P. W., R. G. Bell, and M. C. Nesheim. 1985. Iron deficiency protectsinbred mice against infection with Plasmodium chabaudi. Infect. Immun.50:932–934.

12. Ing, R., P. Gros, and M. M. Stevenson. 2005. Interleukin-15 enhances innateand adaptive immune responses to blood-stage malaria infection in mice.Infect. Immun. 73:3172–3177.

13. Irizarry, R. A., B. Hobbs, F. Collin, Y. D. Beazer-Barclay, K. J. Antonellis, U.Scherf, and T. P. Speed. 2003. Exploration, normalization, and summaries ofhigh density oligonucleotide array probe level data. Biostatistics 4:249–264.

14. Irizarry, R. A., D. Warren, F. Spencer, I. F. Kim, S. Biswal, B. C. Frank, E.Gabrielson, J. G. Garcia, J. Geoghegan, G. Germino, C. Griffin, S. C.Hilmer, E. Hoffman, A. E. Jedlicka, E. Kawasaki, F. Martinez-Murillo, L.Morsberger, H. Lee, D. Petersen, J. Quackenbush, A. Scott, M. Wilson, Y.Yang, S. Q. Ye, and W. Yu. 2005. Multiple-laboratory comparison of mi-croarray platforms. Nat. Methods 2:345–350.

15. Kamis, A. B., and J. B. Ibrahim. 1989. Effects of testosterone on bloodleukocytes in Plasmodium berghei-infected mice. Parasitol. Res. 75:611–613.

16. Kendziorski, C., R. A. Irizarry, K. S. Chen, J. D. Haag, and M. N. Gould.2005. On the utility of pooling biological samples in microarray experiments.Proc. Natl. Acad. Sci. USA 102:4252–4257.

17. Klein, S. L. 2000. The effects of hormones on sex differences in infection:from genes to behavior. Neurosci. Biobehav. Rev. 24:627–638.

18. Klein, S. L. 2004. Hormonal and immunological mechanisms mediating sexdifferences in parasite infection. Parasite Immunol. 26:247–264.

19. Klein, S. L., A. Cernetich, S. Hilmer, E. P. Hoffman, A. L. Scott, and G. E.Glass. 2004. Differential expression of immunoregulatory genes in male andfemale Norway rats following infection with Seoul virus. J. Med. Virol.74:180–190.

20. Kochar, D. K., I. Thanvi, A. Joshi, Shubhakaran, N. Agarwal, and N. Jain.1998. Mortality trends in falciparum malaria: effect of gender difference andpregnancy. J. Assoc. Physicians India 47:774–778.

21. Kriegsfeld, L. J., and R. J. Nelson. 1996. Gonadal and photoperiodic influ-ences on body mass regulation in adult male and female prairie voles. Am. J.Physiol. 270:R1013–R1018.

22. Krucken, J., M. A. Dkhil, J. V. Braun, R. M. Schroetel, M. El-Khadragy, P.Carmeliet, H. Mossmann, and F. Wunderlich. 2005. Testosterone suppressesprotective responses of the liver to blood-stage malaria. Infect. Immun.73:436–443.

23. Landgraf, B., H. Kollaritsch, G. Wiedermann, and W. H. Wernsdorfer. 1994.Parasite density of Plasmodium falciparum malaria in Ghanaian schoolchil-dren: evidence for influence of sex hormones? Trans. R. Soc. Trop. Med.Hyg. 88:73–74.

24. Langhorne, J., F. R. Albano, M. Hensmann, L. Sanni, E. Cadman, C.Voisine, and A. M. Sponaas. 2004. Dendritic cells, pro-inflammatory re-sponses, and antigen presentation in a rodent malaria infection. Immunol.Rev. 201:35–47.

25. Li, C., L. A. Sanni, F. Omer, E. Riley, and J. Langhorne. 2003. Pathology ofPlasmodium chabaudi chabaudi infection and mortality in interleukin-10-deficient mice are ameliorated by anti-tumor necrosis factor alpha and ex-acerbated by anti-transforming growth factor beta antibodies. Infect. Immun.71:4850–4856.

26. Li, C., E. Seixas, and J. Langhorne. 2001. Rodent malarias: the mouse as amodel for understanding immune responses and pathology induced by theerythrocytic stages of the parasite. Med. Microbiol. Immunol. 189:115–126.

27. Loetscher, P., M. Uguccioni, L. Bordoli, M. Baggiolini, B. Moser, C.Chizzolini, and J. M. Dayer. 1998. CCR5 is characteristic of Th1 lympho-cytes. Nature 391:344–345.

28. Martin, R. M., and A. M. Lew. 1998. Is IgG2a a good Th1 marker in mice?Immunol. Today 19:49.

29. McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach,M. Collins, and M. C. Byrne. 2002. CD4� CD25� immunoregulatory T cells:gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:311–323.

30. McKay, L. I., and J. A. Cidlowski. 1999. Molecular control of immune/inflammatory responses: interactions between nuclear factor-�B and steroidreceptor-signaling pathways. Endocrinol. Rev. 20:435–459.

31. Mo, R., J. Chen, A. Grolleau-Julius, H. S. Murphy, B. C. Richardson, andR. L. Yung. 2005. Estrogen regulates CCR gene expression and function inT lymphocytes. J. Immunol. 174:6023–6029.

32. Molineaux, L., and G. Gramiccia. 1979. The Garki project: research on theepidemiology and control of malaria in the Sudan Savannah of West Africa.World Health Organization, Geneva, Switzerland.

33. Morinobu, A., M. Gadina, W. Strober, R. Visconti, A. Fornace, C. Montagna,G. M. Feldman, R. Nishikomori, and J. J. O’Shea. 2002. STAT4 serinephosphorylation is critical for IL-12-induced IFN-gamma production but notfor cell proliferation. Proc. Natl. Acad. Sci. USA 99:12281–12286.

34. Morrow, R. H., A. Kisuule, M. C. Pike, and P. G. Smith. 1976. Burkitt’slymphoma in the Mengo Districts of Uganda: epidemiologic features andtheir relationship to malaria. J. Natl. Cancer Inst. 56:479–483.

35. Omer, F. M., J. B. de Souza, and E. M. Riley. 2003. Differential induction ofTGF-beta regulates proinflammatory cytokine production and determinesthe outcome of lethal and nonlethal Plasmodium yoelii infections. J. Immu-nol. 171:5430–5436.

36. Oppenheimer, S. J. 2001. Iron and its relation to immunity and infectiousdisease. J. Nutr. 131:616S–635S.

37. Owens, I. P. 2002. Ecology and evolution. Sex differences in mortality rate.fsScience 297:2008–2009.

38. Polanczyk, M. J., B. D. Carson, S. Subramanian, M. Afentoulis, A. A.Vandenbark, S. F. Ziegler, and H. Offner. 2004. Cutting edge: estrogendrives expansion of the CD4� CD25� regulatory T-cell compartment. J. Im-munol. 173:2227–2230.

39. Roberts, C. W., W. Walker, and J. Alexander. 2001. Sex-associated hormonesand immunity to protozoan parasites. Clin. Microbiol. Rev. 14:476–488.

40. Sallusto, F., D. Lenig, C. R. Mackay, and A. Lanzavecchia. 1998. Flexibleprograms of chemokine receptor expression on human polarized T helper 1and 2 lymphocytes. J. Exp. Med. 187:875–883.

41. Sam, H., and M. M. Stevenson. 1999. Early IL-12 p70, but not p40, produc-tion by splenic macrophages correlates with host resistance to blood-stagePlasmodium chabaudi AS malaria. Clin. Exp. Immunol. 117:343–349.

42. Sanni, L. A., L. F. Fonseca, and J. Langhorne. 2002. Mouse models forerythrocytic-stage malaria. Methods Mol. Med. 72:57–76.

43. Sapino, A., P. Cassoni, E. Ferrero, M. Bongiovanni, L. Righi, N. Fortunati,P. Crafa, R. Chiarle, and G. Bussolati. 2003. Estrogen receptor alpha is anovel marker expressed by follicular dendritic cells in lymph nodes andtumor-associated lymphoid infiltrates. Am. J. Pathol. 163:1313–1320.

44. Satoskar, A., H. H. Al-Quassi, and J. Alexander. 1998. Sex-determined



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

resistance against Leishmania mexicana is associated with the preferentialinduction of a Th1-like response and IFN-gamma production by female butnot male DBA/2 mice. Immunol. Cell Biol. 76:159–166.

45. Smith, E. C., and A. W. Taylor-Robinson. 2003. Parasite-specific immuno-globulin isotypes during lethal and non-lethal murine malaria infections.Parasitol. Res. 89:26–33.

46. Stevenson, M. M., and E. M. Riley. 2004. Innate immunity to malaria. Nat.Rev. Immunol. 4:169–180.

47. Storey, J. D., and R. Tibshirani. 2003. Statistical significance for genomewidestudies. Proc. Natl. Acad. Sci. USA 100:9440–9445.

48. Su, Z., and M. M. Stevenson. 2000. Central role of endogenous gammainterferon in protective immunity against blood-stage Plasmodium chabaudiAS infection. Infect. Immun. 68:4399–4406.

49. Su, Z., and M. M. Stevenson. 2002. IL-12 is required for antibody-mediatedprotective immunity against blood-stage Plasmodium chabaudi AS malariainfection in mice. J. Immunol. 168:1348–1355.

50. Sullivan, P. S., C. W. Jackson, and T. P. McDonald. 1995. Castration de-creases thrombocytopoiesis and testosterone restores platelet production incastrated BALB/c mice: evidence that testosterone acts on a bipotentialhematopoietic precursor cell. J. Lab. Clin. Med. 125:326–333.

51. Weise, V. H.-J. 1979. Imported cases of malaria into the Federal Republic ofGermany and West Berlin with particular emphasis upon the last 5 years,1973–1977. Bundesgesundheitsblatt 22:1–7. (In German.)

52. Wildling, E., S. Winkler, P. G. Kremsner, C. Brandts, L. Jenne, and W. H.Wernsdorfer. 1995. Malaria epidemiology in the province of Moyen Ogoov,Gabon. Trop. Med. Parasitol. 46:77–82.

53. Wunderlich, F., W. P. Benten, M. Lieberherr, Z. Guo, O. Stamm, C.Wrehlke, C. E. Sekeris, and H. Mossmann. 2002. Testosterone signaling inT cells and macrophages. Steroids 67:535–538.

54. Wunderlich, F., P. Marinovski, W. P. Benten, H. P. Schmitt-Wrede, and H.Mossmann. 1991. Testosterone and other gonadal factor(s) restrict the ef-ficacy of genes controlling resistance to Plasmodium chabaudi malaria. Par-asite Immunol. 13:357–367.

55. Wunderlich, F., W. Maurin, W. P. Benten, and H. P. Schmitt-Wrede. 1993.Testosterone impairs efficacy of protective vaccination against P. chabaudimalaria. Vaccine 11:1097–1099.

56. Zhang, Z., L. Chen, S. Saito, O. Kanagawa, and F. Sendo. 2000. Possiblemodulation by male sex hormone of Th1/Th2 function in protection againstPlasmodium chabaudi chabaudi AS infection in mice. Exp. Parasitol. 96:121–129.

Editor: W. A. Petri, Jr.



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

involvement of gonadal steroids and gamma interferon in...

Documents