Involvement of Gonadal Steroids and Gamma Interferon in Sex Differences in Response to Blood-Stage Malaria Infection
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感染与免疫杂志 2006年第6期
W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205
ABSTRACT
To examine the hormonal and immunological mechanisms that mediate sex differences in susceptibility to malaria infection, intact and gonadectomized (gdx) C57BL/6 mice were inoculated with Plasmodium chabaudi AS-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. Expression microarrays and real-time reverse transcription-PCR revealed that gonadally intact females exhibited higher expression 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 intact males and gdx females, with these differences being most pronounced during peak parasitemia. Intact females also had higher anti-P. chabaudi immunoglobulin G (IgG) and IgG1 responses than either intact males or gdx females. To further examine the effector mechanisms mediating sex differences in response to P. chabaudi infection, responses to infection were compared among male and female wild-type (WT), T-cell-deficient (TCR–/–), B-cell-deficient (μMT), 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 differences being most pronounced among TCR–/–, μMT, and RAG1 mice. Male mice also exhibited more severe weight loss, anemia, and hypothermia, and higher peak parasitemia than females during infection, with WT, RAG1, TCR–/–, and μMT mice exhibiting the most pronounced sexual dimorphism. The absence of IFN- reduced the sex difference in mortality and was more detrimental to females than males. These data suggest that differential transcription and translation of IFN-, that is influenced by estrogens, may mediate sex differences in response to malaria.
INTRODUCTION
Males are more susceptible to many protozoan infections than females and field and laboratory studies link increased susceptibility to infection with circulating steroid hormones (17, 18, 39). One genus of protozoan parasites that causes a pronounced sexual dimorphism in vertebrate hosts is Plasmodium. Among humans, although the incidence of infection is often similar between the sexes (see references 51 and 52), sex differences in the intensity of infection are reported in which men have higher parasitemia than women (23, 32, 52). The observation that P. falciparum (i.e., a human malaria parasite) density increases at puberty in men, but not in women, suggests that circulating sex steroids may influence this outcome (23).
Studies of rodent malarias have confirmed that males are more likely to die after blood-stage malaria infection than are females (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 underlie increased susceptibility to Plasmodium infections in males compared to females. Injection of female mice with high doses of testosterone reduces antibody production, the number of major histocompatibility complex class II cells in the spleen, and the expression of malaria-responsive genes in the liver but does not affect cytokine production (2, 22). Receptors for sex steroids are expressed in various lymphoid tissue cells, as well as in circulating lymphocytes, macrophages, and dendritic cells (8, 39, 43, 53). The binding of sex steroids to their respective steroid receptors directly influences cell signaling pathways, including nuclear factor-B (NF-B), resulting in the differential production of cytokines and chemokines by cells of the immune system (30). Whereas cellular signaling through NF-B induces the expression of immune and inflammatory genes, steroid hormone signaling can antagonize NF-B-mediated responses, resulting in tightly regulated communication between the endocrine and immune systems (30). If sex steroids influence the sexual dimorphism in immune responses to Plasmodium infection, then removal of the sex steroids via gonadectomy may significantly alter immune and inflammatory responses during malaria infection.
Utilization of mice infected with rodent Plasmodium species has been instrumental for characterizing the pathogenesis and immunobiology of blood-stage malaria (46). In mice that are resistant to blood-stage malaria infection, production of interleukin-12 (IL-12), tumor necrosis factor (TNF), and gamma interferon (IFN-) during the acute phase of infection and antibody production during the chronic phase of infection is critical for recovery from Plasmodium chabaudi infection (46). Studies of human and rodent malarias illustrate that proinflammatory immune responses are necessary for the development of protective immunity but must be regulated to prevent pathology (24). The timing and shift from Th1 to Th2 responses during the course of Plasmodium infection is mediated by regulatory responses, including the production of transforming growth factor (TGF-) and IL-10 (25, 35). A majority of the rodent studies characterizing protective immune responses against blood-stage malaria infection have used female mice. Whether the development and timing of protective immune responses during infection differ between males and females and are altered by sex steroid hormones has not been adequately examined.
The primary goal of the present study was to examine sex differences in the pathogenesis and immunobiology of P. chabaudi infection in C57BL/6 mice. Gonadally intact females are more resistant to infection than males; the mechanisms mediating this sex difference remains elusive. We hypothesized that intact females would have reduced parasitemia, anemia, weight loss, and hypothermia during infection compared to males. Because elevated proinflammatory and regulatory T-cell responses during the acute phase of infection and heightened antibody responses during the persistent phase of infection are associated with protection and recovery from infection, we hypothesized that these responses would be elevated among intact females compared to intact males. We further hypothesized that if sex steroids mediate the sexual dimorphism in infection, then the removal of sex steroids via gonadectomy would reverse the sex difference in response to blood-stage malaria infection. Finally, to provide converging evidence for the effector responses that mediate sex-based differences in response to malaria, responses to P. chabaudi challenge were compared among male and female T-cell-deficient (TCR–/–), B-cell deficient (μMT), T- and B-cell-deficient (RAG1), and IFN- deficient (IFN-–/–) mice. Overall, our data reveal that removal of the ovaries (i.e., the primary source of estrogens) significantly alters immune responses during blood-stage malaria infection to a greater extent than removal of the testes, suggesting that sex differences in response to malaria may be estrogen dependent. Our data also illustrate that females rely more heavily on IFN- than do males to overcome malaria infection, indicating that IFN- may play an important role in mediating the sexual dimorphism in resistance to blood-stage malaria infection.
MATERIALS AND METHODS
Animals. Adult (>60-day-old) male (n = 100; 10/time point/treatment) and female (n = 100; 10/time point/treatment) C57BL/6 mice were purchased from the 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 (μMT mice), combined T- and B-cell-deficient (RAG1 mice), and IFN- knockout (IFN–/– mice) mice, all on a C57BL/6 background, were purchased from Jackson Laboratories (Bar Harbor, ME). All animals were housed at five mice/cage in a microisolator room and maintained on a constant light-dark cycle (16 and 8 h). Food and sterile tap water were available ad libitum. The Johns Hopkins Animal Care and Use Committee (protocol MO02H49) and the Johns Hopkins Office of Health, Safety, and Environment (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 maintained in donor BALB/c mice by weekly passage from frozen stock cultures. Parasites were 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 be bilaterally gonadectomized (gdx). Mice were bilaterally gdx under ketamine (80 mg/kg [body mass])-xylazine (8 mg/kg [body mass]) anesthesia (Phoenix Pharmaceutical, 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 (Abbott Laboratories, Chicago, IL); body mass was measured; and blood was collected from the retro-orbital sinus to assess anemia, parasitemia, and anti-P. chabaudi antibody responses. Spleens were dissected and weighed and white blood cells (WBCs) were isolated and used to measure mRNA expression and protein production. Body mass and blood samples also were collected at 5 and 10 days p.i. For experiments with mice with selective deficiencies, following i.p. inoculation with P. chabaudi-infected erythrocytes, parasitemia, body mass, rectal temperature, 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, and stained with a 1:10 dilution of Giemsa stain (Sigma, St. Louis, MO) in 1x phosphate buffer (3 mM potassium phosphate monobasic, 4.7 mM sodium phosphate monobasic [pH 7.1]). Parasites were visualized under a 100x oil immersion 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, tubes were plugged with clay and centrifuged for 15 min at 1,200 rpm, and the red blood cell (RBC) volume was measured relative to the total blood volume.
Body temperature. To monitor body temperature, animals were briefly restrained in a modified 50-ml conical tube, and rectal temperature was measured within 5 s (Physitemp, Clifton, NJ).
WBC isolation. Spleens were placed in sterile RPMI 1640 medium (Mediatech Cellgro, Herndon, VA), and WBCs were recovered by pressing the whole spleen through a 100-μm-pore-size nylon cell strainer. Separated cells were suspended in additional RPMI 1640, and erythrocytes were lysed with 10 ml of erythrocyte lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM Na2+ EDTA [pH 7.2 to 7.4]). Cells were washed twice, resuspended in 10 ml of phosphate-buffered saline (PBS), and WBC counts and viability were determined by using a hemacytometer and trypan blue exclusion.
IFN- ELISA. Viable WBCs were adjusted to 5 x 106 cells/ml in supplemented culture medium (RPMI 1640 with 25 mM HEPES, 10% fetal bovine serum, 2 mM L-glutamine, 1% penicillin-streptomycin, 0.2 mM 2-mercaptoethanol). Plates were incubated at 37°C with 5% CO2 for 72 h. The cell supernatant was removed from each well, centrifuged for 5 min at 5,000 rpm, transferred into sterile tubes, and stored at –80°C. IFN- concentrations were assayed by enzyme-linked immunosorbent assay (ELISA) by diluting samples to 1:10 and using the manufacturer's protocols for the OptEIA IFN- kit (BD Pharmingen, San Diego, CA).
Anti-P. chabaudi ELISA. Lysate of P. chabaudi antigen was used as a capture antigen, and the protein concentration was estimated by using a bicinchoninic acid protein assay (Pierce, Rockford, IL). Microtiter plates were coated overnight at 4°C in carbonate buffer with 5 μg of P. chabaudi antigen/ml for immunoglobulin G (IgG) or 20 μg 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) for IgG 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 and 1:50 for IgG1 and IgG2c in PBS (sera dilutions were based on optimization assays). 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 PBS for 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 optical density was measured at 450 nm, and the samples were expressed as a percentage of the positive control run on the same microtiter plate.
Microarrays. Viable WBCs were adjusted to 5 x 106 cells/ml by dilution with PBS and stored in liquid nitrogen. For RNA extraction, TRIzol LS (Invitrogen, Carlsbad, CA) was added to cells, followed by high-speed disruption in a FastPrep 120 Instrument (Q-Biogene, Irvine, CA) at speed 5 for 15 s. Homogenates were subsequently processed according to the manufacturer's protocol (Invitrogen). RNA pellets were resuspended in nuclease-free water. The quantitation of RNA was performed by using a Beckman DU640 spectrophotometer, and an RNA quality assessment was determined by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100. Equivalent aliquots (2.5 μg) of RNA from three animals/treatment group were pooled, and three separate pools were processed on separate Affymetrix GeneChips (n = 3 GeneChips/time point/treatment group) (16). The variability among the three GeneChips, within each treatment group at each time point ranged from 0.83 to 2.25%.
The processing of RNA for GeneChip analysis was in accordance with the methods described in the Affymetrix GeneChip Expression Analysis Technical Manual, revision three, as previously reported (14). Hybridization cocktails were prepared as recommended prior to pipetting into the Affymetrix GeneChips (Murine Genome MOE430A). Hybridization and the signal amplification protocol for washing and staining of eukaryotic targets were performed as previously described (14). The arrays were scanned at an emission wavelength of 570 nm at 2.5-mm resolution in the GCS3000 laser scanner (Affymetrix). The intensity of the hybridization for each probe pair was computed by GCOS 1.1 software. For more detailed methods, please refer to the Web site of the Malaria Research Institute Gene Array Core Facility at the Johns Hopkins Bloomberg School of Public Health (http://jhmmi.jhsph.edu/). Primary analysis of microarrays consisted of a quality assessment of the hybridization for each sample. The ratios of signal for probe sets at the 5' and 3' regions of the housekeeping genes were calculated and monitored as an indication of the transcript quality for each sample.
Real-time RT-PCR for microarray validation. Custom primer and probe sets were generated for each target gene by using Primer Express 2.0 software (Applied Biosystems, Foster City, CA). Reverse transcription (RT) of 1.5 μg of RNA from each individual animal sample (n = 8 to 10/time point/treatment group) was conducted with oligo(dT)16 primers and according to the manufacturer's protocol for the SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen). cDNA was amplified in a reaction mixture containing 50 μM concentrations of the primers, a 250 μM concentration of the probe, and the TaqMan Universal PCR mix according to the manufacturer's protocol (Applied Biosystems). All reactions were run in optical 96-well plates using the Prism 7000 Sequence Detection System (Applied Biosystems). Serial dilutions of pooled cDNA from randomly selected animals from each treatment group at each time point 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 were reported relative to the values from same-treatment uninfected control animals.
Statistical analyses. The proportion of animals that survived infection was compared among experimental groups by chi-square analyses. Survival curves were compared between sexes by using Cox Hazard regression analyses and log-rank analyses. Parasitemia, anemia, body temperature, and body mass changes were analyzed by using mixed analyses of variance (ANOVAs), with one within subjects variable (days p.i.) and one between subjects variable (treatment group). Antibody responses and splenomegaly were analyzed by using two-way ANOVAs with two between subjects variable (day p.i. and treatment group). IFN- concentrations were assessed by using a one-way ANOVA. Significant interactions were further analyzed by using planned comparisons or the Tukey method for pairwise multiple comparisons. The combined contribution of parasitemia, anemia, weight loss, and hypothermia to mortality was investigated by using multiple linear regression. Correlations among dependent variables were assessed by using Pearson product moment correlational analyses. Mean differences were considered statistically significant if P was <0.05.
For microarray analyses, data were preprocessed by using GC-RMA in GeneSpring 7.2 (13). Gene expression patterns for each gene from infected animals were normalized to the expression levels from same-treatment uninfected control (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 compare gene expression patterns between intact males and females and between gdx and intact animals of the same sex, and mean differences were considered statistically significant if P was <0.05. Because thousands of statistical tests were performed on 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 that for the genes that had a statistically significant difference, the false discovery rates 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 the NCBI Gene Expression Omnibus (GEO accession GSE4324; www.ncbi.nlm.nih.gov/geo/).
RESULTS
Intact males are more likely to die from infection and recover more slowly from infection-induced weight loss and anemia than intact females. To determine whether responses to blood-stage malaria differed between the sexes and were affected by the presence or absence of gonadal steroids, intact and gdx C57BL/6 mice were inoculated with P. chabaudi AS-infected erythrocytes, and body mass, anemia, parasitemia, antibody responses, IFN- concentrations, and expression levels of immune-related genes were monitored. Significantly more intact C57BL/6 males (7 of 25) died from P. chabaudi infection than gdx males (0 of 20), intact females (1 of 24), and gdx females (1 of 21) (P = 0.005; Fig. 1A). Death from P. chabaudi infection occurred between days 8 and 10 p.i. Levels of parasitemia peaked 7 days after inoculation with P. chabaudi for all animals (P < 0.0001; Fig. 1B). The peak parasitemia was marginally higher for gdx males than for intact males, intact females, 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 infected animals relative to the responses from uninfected same sex and treatment controls. After inoculation with P. chabaudi, body mass decreased significantly through day 10 p.i. and returned to baseline by day 21 p.i. for all animals (P < 0.0001; Fig. 1C). The body mass of intact females, however, returned to baseline faster than for intact males and gdx females, as noted by the rapid weight gain among intact females 14 and 21 days p.i. (P < 0.05; Fig. 1C). After inoculation with P. chabaudi, the percentage 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 from anemia faster than intact males and gdx females, as indicated by the rapid replacement of RBC in intact females by 14 days p.i. (P = 0.06; Fig. 1D). Over the course of infection, the spleen mass increased, whereas the WBC counts decreased, for all animals by 14 and 21 days p.i. (P < 0.001 in each case; data not shown). There were no sex differences or effects of gonadectomy 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 males and females and is altered by gonadectomy. Expression microarrays and real-time RT-PCR were used to measure changes 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 genes arrayed on the mouse MOE430A GeneChip (Affymetrix), 492 genes showed differential expression levels between intact males and intact females, 45 genes were differentially expressed between intact males and gdx males, and 3 genes differed between intact females and gdx females. Because baseline differences in gene expression were observed, gene expression patterns for each gene from infected animals were normalized to the expression levels from same-treatment uninfected control (i.e., day 0) animals (i.e., per gene normalization) (19), which enabled sex or treatment differences in the expression levels of genes 3 to 14 days p.i. to be attributed to infection.
During infection (days 3 to 14 p.i.), 5,182 genes showed significant differences in the relative expression intensity between the sexes, 4,360 genes differed between intact males and gdx males, and 7,013 genes differed between intact females and gdx females (P < 0.05 in each case). Using GO Biological Processes descriptions, we identified 74 unique genes with associated immune function that had expression levels that differed significantly between intact males and intact females during infection (P < 0.05; Fig. 2A and Table S1 in the supplemental material); 53 unique immune-related genes that differed significantly between intact males and gdx males during infection (P < 0.05; Fig. 2B and Table S2 in the supplemental material), and 60 unique immune-related genes that differed significantly between intact females and gdx females during P. chabaudi infection (P < 0.05; Fig. 2C and Table S3 in the supplemental material). Differential expression of genes during P. chabaudi infection was observed 3, 7, and 14 days p.i. (Tables S1 to S3 in the supplemental material); differences in gene expression levels among the groups of mice, however, were most pronounced during peak parasitemia (i.e., day 7 p.i.; Fig. 2).
Among intact mice, males and females exhibited sexually dimorphic expression of several genes associated with innate immunity, antigen presentation, cell adhesion, cytokines, iron metabolism, apoptosis, cellular proliferation, complement, and antibody production during P. chabaudi infection (P < 0.05; Fig. 2A and Table S1 in the supplemental material). Of specific interest, genes involved in the production of IFN- and the development of Th1 immunity were consistently upregulated among intact female mice during peak parasitemia. For example, CCL3 (i.e., MIP-1), CXCL10 (i.e., IP-10), CCR5, IFN-, growth arrest- and DNA-damage-inducible 45 (Gadd45), IL-12R2, and IL-15R, as well as IFN//-inducible genes (e.g., IFN--inducible protein 16 [Ifi16] and IFN-induced protein with tetratricopeptide repeats 2 [Ifit2]) had significantly higher expression levels in intact females than intact males during peak parasitemia (Fig. 2A and Table S1 in the supplemental material). In particular, the expression of IFN- was sixfold higher in intact females than in intact males during peak parasitemia (Fig. 3C). In addition, the expression of regulatory T-cell genes, including IL-10 and TNFR superfamily member 4 (Tnfrsf4; OX-40), was twofold higher among intact female than intact male mice during peak parasitemia (Fig. 4A and B). IFN- inhibits iron metabolism in WBCs (4) and several genes associated with iron regulation, including lactotransferrin (Ltf) and transferrin receptor 2 (TrfR2), were significantly downregulated among intact females compared to intact males (P < 0.05; Fig. 5A and B.
During infection, gonadectomy of male and female mice significantly altered the expression of immune-related genes relative to their intact counterparts. Gonadectomy of male and female mice significantly altered the expression of genes associated with innate immunity, chemokines, cytokines, antigen presention, regulatory responses, cellular proliferation, apoptosis, and signal transduction compared to their intact counterparts (P < 0.05; Fig. 2B and C and Tables S2 and S3 in the supplemental material). Gonadectomy of male mice increased the expression of IL-15R, Gadd45, IFN-induced transmembrane protein 3-like (Ifitm3l), IFN--inducible Puma-g (Gpr109b), Myd88, CCL3, IFN-related developmental regulator 1 (Ifrd1), and TGF- induced (Tgfbi) compared to intact males (Fig. 2B and Table S2 in the supplemental material). Gonadectomy of female mice significantly suppressed the expression of several genes associated with Th1 immunity and regulatory T-cell responses, including Ifrd1, TGF--inducible early growth response 1 (Tieg1) CCR5, IFN-, Gadd45, IL-12R2, and IL-15R, during infection (Fig. 2C and Table S3 in the supplemental material).
Because of the potential problem of false positives associated with microarray analyses, several genes of interest were validated by using real-time RT-PCR. Real-time RT-PCR also was used to further examine gene expression profiles at 21 days p.i. The expression patterns of IL-12R2, IFN-, Gadd45, IL-10, Tnfrsf4, Ltf, TrfR, and -actin in WBCs from individual animals were examined, and the expression levels of each gene from infected animals were analyzed relative to the expression levels of each gene from uninfected same-treatment control animals. The expression of the housekeeping gene -actin was similar among intact and gdx males and females days 0 to 21 p.i. (P > 0.05). Consistent with our microarray results, the expression of genes associated with the synthesis of IFN-, including IL-12R, Gadd45, and IFN-, was higher among intact females than intact males, gdx males, and gdx females 7 days p.i. (P < 0.01 in each case; Fig. 3). Although microarray analyses revealed that gdx males had a higher expression of 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 isolated from intact females produced more IFN- than did WBCs isolated 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 highest expression 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 females had higher expression of regulatory genes, including IL-10 and Tnfrsf4, than intact males during peak parasitemia (P < 0.001 in each case; Fig. 4); real-time RT-PCR results further indicated that gonadectomy of females significantly suppressed the expression of IL-10 and Tnfrsf4 and gonadectomy of males enhanced the expression of IL-10 at day 7 p.i. (Fig. 4C and D). Microarray and real-time RT-PCR analyses consistently illustrated that the expression of genes associated with iron homeostasis, including Ltf and TrfR, was higher among intact males than among intact females 14 days p.i. and that gonadectomy of male mice significantly reduced the expression of the TrfR (P < 0.001 in each case; Fig. 5).
During the recovery phase of infection, intact females have higher antibody responses than either intact males or gdx females. To eliminate the erythrocytic stage of Plasmodium parasites, both humoral and cell-mediated effector mechanisms are required. Anti-P. chabaudi IgG increased over time for all animals and peaked at 14 to 21 days p.i. (P < 0.001; Fig. 6A). Anti-P. chabaudi IgG antibody responses, however, were higher among intact females than among intact males at 14 to 21 days p.i; gonadectomy of female mice reversed this sex difference by 21 days p.i. (P < 0.01; Fig. 6A).
To assess whether different subsets of helper T cells were activated by males and females in response to infection, subclasses of IgG were examined. Anti-P. chabaudi IgG1 responses increased over the course of infection and were highest at 14 to 21 days p.i. for all animals (P < 0.001; Fig. 6B). Anti-P. chabaudi IgG1 responses were significantly higher among intact females than intact males at 14 days p.i. (P < 0.01; Fig. 6B). C57BL/6 mice do not possess the gene for IgG2a but express a novel isotype, IgG2c, that is indicative of a Th1-mediated response (28). Anti-P. chabaudi IgG2c responses peaked 14 to 21 days p.i. for all animals (P < 0.001; Fig. 6C) but did not differ significantly among the experimental groups of mice (P > 0.05; Fig. 6C).
Utilization of immunodeficient mice reveals that innate immunity may mediate sexually dimorphic responses to P. chabaudi infection. To determine the roles of innate and acquired immune responses, as well as the specific effects of IFN-, as mediators of sex differences in P. chabaudi infection, we inoculated adult WT, TCR–/–, μMT, RAG1, and IFN-–/– mice with P. chabaudi parasites and monitored sex differences in parasitemia, body mass, rectal temperature, anemia, and mortality. Peak parasitemia occurred for all mice at 7 days p.i. and was followed by a period of reduced parasitemia in WT, RAG1, and μMT mice (Fig. 7). In contrast, among TCR–/– 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, μMT, and IFN-–/– mice compared to their female counterparts (P < 0.05 in each case; Fig. 7). Among TCR–/– mice, although peak parasitemia was similar between males and females, males had a higher proportion of parasitized erythrocytes at 10 days p.i. (because only one TCR–/– male was still alive days 14 to 21 p.i, statistical comparisons could not be made at these time points) (P < 0.05; Fig. 7). Peak parasitemia was similarly high for both male and female RAG1 mice (P > 0.05; Fig. 7).
A Cox Hazard regression analysis across all strains of mice revealed that males were 3.5 times (95% confidence interval of 2.13 to 5.71) more likely to die from blood-stage malaria infection than were females. Based on log-rank analyses, survival was significantly prolonged for female compared to male RAG1, TCR–/–, and μMT mice (P < 0.001 in each case; Fig. 7). As a result, the average day of death was significantly earlier for male than female RAG1 (P < 0.01 [males, 8 days p.i.; females, 14 days p.i.]), TCR–/– (P < 0.001 [males, 10 days p.i.; females, 21 days p.i.]), and μMT (P < 0.01 [males, 8 days 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 male IFN-–/– mice began dying significantly sooner after inoculation with P. chabaudi than did females (P < 0.05; Fig. 7). Among male mice, deletion of T cells, B cells, or both lymphocyte populations significantly increased the likelihood of dying from blood-stage malaria infection compared to WT mice (P < 0.001 in each case). Among female mice, the absence of IFN-, B cells, or both T and B cells significantly increased mortality from P. chabaudi infection compared to WT 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 had less severe hypothermia during infection and generally returned to baseline faster than males (P < 0.05 in each case; Fig. 8) . Among RAG1 and TCR–/– mice, males lost more weight than females by 3 to 7 days p.i. (P > 0.05; Fig. 8). Female RAG1, μMT, and TCR–/– mice consistently developed less severe hypothermia than their male counterparts (P < 0.01 in each case; Fig. 8). Male and female RAG1, μMT, and TCR–/– mice exhibited a similar decrease in the percentage of RBCs through day 7 p.i. (P > 0.05 in each case; Fig. 8). Among IFN-–/– mice, females exhibited an initial decrease in body mass, the percentage of RBCs, and body temperature that was followed by an early return to baseline by day 10 p.i. and a subsequent relapse 14 days p.i., resulting in a pattern that was significantly different from that of the WT female mice (P < 0.01 in each case). Conversely, male IFN-–/– mice demonstrated a more consistent decline in body mass, percentage of RBC, and body temperature through day 10 p.i., followed by a return to baseline by day 14 p.i., which was not significantly different from WT males (P > 0.05 in each case). The dimorphic response of IFN-–/– mice to infection resulted in IFN-–/– males having more severe weight loss, anemia, and hypothermia than IFN-–/– females at 10 days p.i. and IFN-–/– females exhibiting increased morbidity 14 days p.i. (P < 0.001 in each case; Fig. 8).
Multiple linear regression analyses revealed that at 7 days p.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 hypothermia, but not parasitemia, anemia, or weight loss, in both males (P < 0.001) and females (P < 0.001), regardless of their immunodeficiency.
DISCUSSION
In the present series of studies, gonadally intact WT males were more likely than intact WT females to die after P. chabaudi infection, exhibit slower recovery from infection-associated weight loss, hypothermia, and anemia, have reduced IFN--associated gene expression and IFN- production during peak parasitemia, and produce less antibody during the recovery phase of infection. Gonadectomy of male and female WT mice altered these sex-associated differences, suggesting that sex steroid hormone, in particular androgens and estrogens, may modulate immune responses to infection.
Infection of C57BL/6 mice with P. chabaudi causes transient anemia, weight loss, and hypothermia. These sequelae, combined with elevated parasitemia, are associated with increased mortality from blood-stage malaria infection (42). In the present study, elevated parasitemia at 7 days p.i. and severe hypothermia at 10 days p.i. were significant predictors of death from P. chabaudi in both male and female mice. Susceptibility to P. chabaudi also is associated with low inflammatory responses during the acute phase of infection (46). We observed that individuals with the highest concentrations of IFN- tended to have the lowest levels of parasitemia, which may contribute to the increased likelihood of surviving infection among females compared to males.
Characterization of the immunological causes of sex differences in Plasmodium infection using immunodeficient mice revealed that sex differences in parasitemia, morbidity (as measured by changes body mass, RBC loss, and development of hypothermia), and mortality were still apparent in the absence of T cells, B cells, or both cell populations. Resistance to blood-stage malaria was compromised in immunodeficient male and female mice relative to their WT counterparts, since both male and female mice deficient in T cells, B cells, or both cell populations experienced chronic parasitemia and increased mortality compared to WT mice. Thus, acquired immunity plays a critical role in mediating the clearance of parasites and recovery from infection. These immunodeficiencies, however, were consistently more detrimental to males than to females because the morbidity and mortality rates, as well as the levels of parasitemia, were still higher in RAG1, TCR–/–, and μMT male mice than in female mice. Our data suggest an important role of innate immunity, including the activity of macrophages, dendritic cells, and NK cells (all of which are functional in RAG1, TCR–/–, and μMT mice), in mediating the sexual dimorphism in susceptibility to P. chabaudi.
To further assess immunological differences in response to malaria infection, microarrays and real-time RT-PCR were used to map the expression levels of immune-related genes in WBCs over the course of P. chabaudi infection. Previous studies illustrate that the administration of pharmacological doses of testosterone suppresses the expression of immune-related genes, including Gadd45 and CXCL10, in the livers, but has no effect on gene expression in the spleens, of female mice infected with P. chabaudi and tested at day 8 p.i. only (22). Female mice injected with high doses of testosterone also exhibit reduced antibody production and expression of major histocompatibility complex class II on spleen cells but do not show altered cytokine production in the spleen during the acute phase of infection compared to intact female mice injected with vehicle (2). Whether immunological parameters differ between males and females during infection has not been systematically examined over the course of infection prior to the present study. We demonstrate that females respond to P. chabaudi infection with a heightened IFN--mediated response during the acute phase of infection, an elevated antibody response during the chronic phase of infection, and reduced manifestation of disease compared to males. We also demonstrate that gonadectomy of females reverses the female-typic immune response to infection, suggesting that estradiol may play a more central role than previously assumed. As a result, our data demonstrate that, in addition to the role played by testosterone, estrogens may play a pivotal role in immune modulation of malaria infection.
The timing of Th1 and Th2 responses is critical for protection against many Plasmodium parasites (26, 46). Elevated Th1-mediated responses during the acute phase of infection are required to reduce peak parasitemia, and activation of Th2-mediated responses during the chronic phase of infection is required to clear parasites (46). Macrophage and dendritic cell-derived IL-12 is important for the development of Th1 phenotypic responses, such as the production of IFN-. Mice that are resistant to blood-stage Plasmodium infection produce more splenic IL-12 and IFN- prior to and during peak parasitemia than do susceptible mice during P. chabaudi infection (41, 48, 49). In the present study, intact females exhibited higher expression of IFN--associated genes, including IL-12R2 and IFN-, and produced more IFN- than did intact males during peak parasitemia. Genes involved in the synthesis of IFN-, including Gadd45, which induces phosphorylation of STAT4 by the p38 pathway (33), showed a similar pattern of expression. IL-15 promotes Th-1-dependent immunity against P. chabaudi infection (12), and the expression of IL-15R was higher on WBCs from intact females than from intact males and was reversed by gonadectomy of both sexes during peak parasitemia. Several chemokines, including CCL3 and CXCL10, that preferentially attract Th1 cells (40), were upregulated in the WBCs from intact females. The chemokine receptor CCR5, which binds CCL3, is expressed at high levels on Th1 cells (27), and is regulated by estrogens (31), also was upregulated on WBCs from intact females. The removal of estrogens via gonadectomy significantly reduced the expression of these Th1-related genes and IFN- production in a manner similar to the reported effects of estradiol on IFN- responses against Leishmania mexicana (44).
If IFN- is the primary mediator of sex differences in response to P. chabaudi, then elimination of IFN- should reduce the sexual dimorphism. Although IFN-–/– males had higher peak parasitemia than did conspecific females, similar proportions of male and female IFN-–/– mice died after infection, providing further evidence that sex differences during blood-stage malaria infection may be influenced by the production of IFN-. The removal of IFN- was consistently more detrimental to females than to males, as illustrated by the increased mortality rates and relapse in body mass loss, RBC loss, and hypothermia in female IFN-–/– compared to female WT mice. It should be noted, however, that our observation of a similar rate of survival among male and female IFN-–/– mice after P. chabaudi AS inoculation is in contrast to previous data 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 mediator of sex differences in response to blood-stage malaria infection.
After peak parasitemia, Th1 responses begin to decline and Th2 phenotypic responses increase in mice that are resistant to Plasmodium infection (46). Intact females had significantly higher anti-P. chabaudi IgG1 responses than males 14 days after infection, suggesting that Th2 responses may be elevated among intact females during the recovery phase of infection. Among intact females, elevated IFN- during the acute phase of infection may induce increased antibody production during later phases of malaria infection (49). Overall, intact females developed higher antibody responses than intact males, and removal of the ovaries and, hence, the primary source of estrogens, significantly suppressed antibody production. Consequently, estrogens potentiate (10) and the administration of testosterone to female mice reduces (2) antibody production by B cells. Although intact females produced higher antibody responses against P. chabaudi, immunoglobulin gene transcripts were elevated in WBCs from intact males, suggesting that there are posttranscriptional differences between the sexes. Although antibody production was suppressed in intact WT males, the removal of B cells in μMT mice did not abolish the sexual dimorphism in response to P. chabaudi infection, suggesting that B cells may not be critical mediators of sex differences in malaria infection.
Regulatory responses, including the production of TGF- and IL-10, mediate the timing and shift from Th1 to Th2 responses and are critical for mitigating pathology caused by prolonged elevation of inflammatory responses during the course of malaria infection (25, 35). Although sex differences in TGF- expression were not observed, IL-10 expression was higher in WBCs from intact females than from intact males, and gonadectomy of females reduced, whereas gonadectomy of males increased, the transcription of IL-10. Whether the differential expression of IL-10 is mediated by macrophages, T cells, or both requires additional investigation. The expression of several genes associated with activated CD4+ CD25+ T cells (29), including Tnfrsf4, Ltb, CCL3, and Gadd45, was higher among intact females than among intact males and was reduced by gonadectomy of female mice in the present study. Estrogens can augment the expansion of CD4+ CD25+ T cells in mice (38). Previous data illustrate that administration of high doses of testosterone to intact females has no effect on production of IFN- or IL-10 (2); thus, sex differences in cytokine and possibly chemokine production during malaria infection are likely mediated by estrogens and not androgens, as previously hypothesized.
In addition to being a key player in Th1 immunity against malaria, IFN- plays a critical role in iron metabolism in WBCs. IFN- reduces iron uptake by macrophages both indirectly through nitric oxide and iron-regulatory proteins and directly by causing downregulation of iron receptors, such as the TrfR (4). In the present study, the transcription of TrfR was downregulated among intact females and gdx males compared to intact males. Elevated IFN- production among intact females may be one mechanism mediating reduced expression of the TrfR. Whether IFN- alters TrfR expression directly or through NO-mediated degradation of iron regulatory proteins requires additional investigation. In the present study, inducible NO synthase expression did not differ between the sexes, suggesting that the direct effects of IFN- on TrfR expression may be involved. Although iron supplementation is beneficial for resolving anemia, iron chelation, which may be regulated by IFN-, effectively reduces the incidence and severity of P. falciparum infection in children (9, 36). In mice, iron-supplemented diets increase parasitemia and mortality following P. chabaudi infection to a greater extent among males than females (11), suggesting that dimorphic iron regulation may contribute to differential responses to infection.
Although sex differences in response to malaria infection are reported among both adults and children, little is known about the mechanisms mediating these differences or whether these sex differences will affect responses to drug treatments or vaccines. Men and women differ in disease manifestations after infection; men are more likely to develop lymphomas, whereas women are more likely to develop anemia, after infection (5, 6, 34). In general, most studies of malaria in human populations do not distinguish between the responses of males and females and, thus, the prevalence of sex differences may be underreported (1). A few studies do, however, clearly illustrate that men are more susceptible than women. For example, several studies indicate that men tend to have higher parasitemia than women (23, 32, 52). In a recent prospective study of imported malaria cases in France, men reported more severe symptoms of malaria infection (e.g., chills, fever, and low platelet counts) than did women (7). Among Ghanaian school children, although the prevalence of P. falciparum infection does not differ between the sexes, the parasite density is significantly higher for boys than girls around puberty (i.e., from ages 8 to 16), suggesting that circulating sex steroids may influence this outcome (23). Although significantly more men die from infectious diseases than women (37), sex differences in mortality from Plasmodium infection are not consistently reported. There are contradictory findings from a study in India, wherein mortality rates were reportedly higher in women than men after infection with P. falciparum (20).
Our studies with a rodent malaria model indicate that males and females respond differently to malaria infection. Reduced rates of parasitemia and faster recovery from hypothermia likely contribute to reduced death rates among females compared to males. The immunological mechanisms that underlie the dimorphism in morbidity and mortality likely involve the heightened IFN- and regulatory T-cell response observed in female mice. These data make a compelling case for the reevaluation of previous epidemiological studies as well as development of prospective studies that are designed to systematically test the hypothesis that males and females differ in their responses to malaria infection. Although sex differences in mortality and parasitemia following Plasmodium infection are reported among rodents, the immunological mechanisms mediating these differences have not been adequately examined. The data from the present study provide candidate immunological pathways that differ between the sexes and that are significantly altered by the absence of estrogens during blood-stage malaria infection.
ACKNOWLEDGMENTS
We thank Mary Stevenson and Mifong Tam for providing the initial stock of P. chabaudi parasites and for assistance with assay development. We also thank Marie Diener-West and Gregory Glass for statistical advice and Judith Easterbrook, Autumn Girouard, Michele Hannah, Peter Klein, and Brandon Zonker for technical assistance.
Financial support for this study was provided by The Johns Hopkins Malaria Research Institute.
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.
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ABSTRACT
To examine the hormonal and immunological mechanisms that mediate sex differences in susceptibility to malaria infection, intact and gonadectomized (gdx) C57BL/6 mice were inoculated with Plasmodium chabaudi AS-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. Expression microarrays and real-time reverse transcription-PCR revealed that gonadally intact females exhibited higher expression 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 intact males and gdx females, with these differences being most pronounced during peak parasitemia. Intact females also had higher anti-P. chabaudi immunoglobulin G (IgG) and IgG1 responses than either intact males or gdx females. To further examine the effector mechanisms mediating sex differences in response to P. chabaudi infection, responses to infection were compared among male and female wild-type (WT), T-cell-deficient (TCR–/–), B-cell-deficient (μMT), 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 differences being most pronounced among TCR–/–, μMT, and RAG1 mice. Male mice also exhibited more severe weight loss, anemia, and hypothermia, and higher peak parasitemia than females during infection, with WT, RAG1, TCR–/–, and μMT mice exhibiting the most pronounced sexual dimorphism. The absence of IFN- reduced the sex difference in mortality and was more detrimental to females than males. These data suggest that differential transcription and translation of IFN-, that is influenced by estrogens, may mediate sex differences in response to malaria.
INTRODUCTION
Males are more susceptible to many protozoan infections than females and field and laboratory studies link increased susceptibility to infection with circulating steroid hormones (17, 18, 39). One genus of protozoan parasites that causes a pronounced sexual dimorphism in vertebrate hosts is Plasmodium. Among humans, although the incidence of infection is often similar between the sexes (see references 51 and 52), sex differences in the intensity of infection are reported in which men have higher parasitemia than women (23, 32, 52). The observation that P. falciparum (i.e., a human malaria parasite) density increases at puberty in men, but not in women, suggests that circulating sex steroids may influence this outcome (23).
Studies of rodent malarias have confirmed that males are more likely to die after blood-stage malaria infection than are females (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 underlie increased susceptibility to Plasmodium infections in males compared to females. Injection of female mice with high doses of testosterone reduces antibody production, the number of major histocompatibility complex class II cells in the spleen, and the expression of malaria-responsive genes in the liver but does not affect cytokine production (2, 22). Receptors for sex steroids are expressed in various lymphoid tissue cells, as well as in circulating lymphocytes, macrophages, and dendritic cells (8, 39, 43, 53). The binding of sex steroids to their respective steroid receptors directly influences cell signaling pathways, including nuclear factor-B (NF-B), resulting in the differential production of cytokines and chemokines by cells of the immune system (30). Whereas cellular signaling through NF-B induces the expression of immune and inflammatory genes, steroid hormone signaling can antagonize NF-B-mediated responses, resulting in tightly regulated communication between the endocrine and immune systems (30). If sex steroids influence the sexual dimorphism in immune responses to Plasmodium infection, then removal of the sex steroids via gonadectomy may significantly alter immune and inflammatory responses during malaria infection.
Utilization of mice infected with rodent Plasmodium species has been instrumental for characterizing the pathogenesis and immunobiology of blood-stage malaria (46). In mice that are resistant to blood-stage malaria infection, production of interleukin-12 (IL-12), tumor necrosis factor (TNF), and gamma interferon (IFN-) during the acute phase of infection and antibody production during the chronic phase of infection is critical for recovery from Plasmodium chabaudi infection (46). Studies of human and rodent malarias illustrate that proinflammatory immune responses are necessary for the development of protective immunity but must be regulated to prevent pathology (24). The timing and shift from Th1 to Th2 responses during the course of Plasmodium infection is mediated by regulatory responses, including the production of transforming growth factor (TGF-) and IL-10 (25, 35). A majority of the rodent studies characterizing protective immune responses against blood-stage malaria infection have used female mice. Whether the development and timing of protective immune responses during infection differ between males and females and are altered by sex steroid hormones has not been adequately examined.
The primary goal of the present study was to examine sex differences in the pathogenesis and immunobiology of P. chabaudi infection in C57BL/6 mice. Gonadally intact females are more resistant to infection than males; the mechanisms mediating this sex difference remains elusive. We hypothesized that intact females would have reduced parasitemia, anemia, weight loss, and hypothermia during infection compared to males. Because elevated proinflammatory and regulatory T-cell responses during the acute phase of infection and heightened antibody responses during the persistent phase of infection are associated with protection and recovery from infection, we hypothesized that these responses would be elevated among intact females compared to intact males. We further hypothesized that if sex steroids mediate the sexual dimorphism in infection, then the removal of sex steroids via gonadectomy would reverse the sex difference in response to blood-stage malaria infection. Finally, to provide converging evidence for the effector responses that mediate sex-based differences in response to malaria, responses to P. chabaudi challenge were compared among male and female T-cell-deficient (TCR–/–), B-cell deficient (μMT), T- and B-cell-deficient (RAG1), and IFN- deficient (IFN-–/–) mice. Overall, our data reveal that removal of the ovaries (i.e., the primary source of estrogens) significantly alters immune responses during blood-stage malaria infection to a greater extent than removal of the testes, suggesting that sex differences in response to malaria may be estrogen dependent. Our data also illustrate that females rely more heavily on IFN- than do males to overcome malaria infection, indicating that IFN- may play an important role in mediating the sexual dimorphism in resistance to blood-stage malaria infection.
MATERIALS AND METHODS
Animals. Adult (>60-day-old) male (n = 100; 10/time point/treatment) and female (n = 100; 10/time point/treatment) C57BL/6 mice were purchased from the 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 (μMT mice), combined T- and B-cell-deficient (RAG1 mice), and IFN- knockout (IFN–/– mice) mice, all on a C57BL/6 background, were purchased from Jackson Laboratories (Bar Harbor, ME). All animals were housed at five mice/cage in a microisolator room and maintained on a constant light-dark cycle (16 and 8 h). Food and sterile tap water were available ad libitum. The Johns Hopkins Animal Care and Use Committee (protocol MO02H49) and the Johns Hopkins Office of Health, Safety, and Environment (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 maintained in donor BALB/c mice by weekly passage from frozen stock cultures. Parasites were 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 be bilaterally gonadectomized (gdx). Mice were bilaterally gdx under ketamine (80 mg/kg [body mass])-xylazine (8 mg/kg [body mass]) anesthesia (Phoenix Pharmaceutical, 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 (Abbott Laboratories, Chicago, IL); body mass was measured; and blood was collected from the retro-orbital sinus to assess anemia, parasitemia, and anti-P. chabaudi antibody responses. Spleens were dissected and weighed and white blood cells (WBCs) were isolated and used to measure mRNA expression and protein production. Body mass and blood samples also were collected at 5 and 10 days p.i. For experiments with mice with selective deficiencies, following i.p. inoculation with P. chabaudi-infected erythrocytes, parasitemia, body mass, rectal temperature, 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, and stained with a 1:10 dilution of Giemsa stain (Sigma, St. Louis, MO) in 1x phosphate buffer (3 mM potassium phosphate monobasic, 4.7 mM sodium phosphate monobasic [pH 7.1]). Parasites were visualized under a 100x oil immersion 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, tubes were plugged with clay and centrifuged for 15 min at 1,200 rpm, and the red blood cell (RBC) volume was measured relative to the total blood volume.
Body temperature. To monitor body temperature, animals were briefly restrained in a modified 50-ml conical tube, and rectal temperature was measured within 5 s (Physitemp, Clifton, NJ).
WBC isolation. Spleens were placed in sterile RPMI 1640 medium (Mediatech Cellgro, Herndon, VA), and WBCs were recovered by pressing the whole spleen through a 100-μm-pore-size nylon cell strainer. Separated cells were suspended in additional RPMI 1640, and erythrocytes were lysed with 10 ml of erythrocyte lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM Na2+ EDTA [pH 7.2 to 7.4]). Cells were washed twice, resuspended in 10 ml of phosphate-buffered saline (PBS), and WBC counts and viability were determined by using a hemacytometer and trypan blue exclusion.
IFN- ELISA. Viable WBCs were adjusted to 5 x 106 cells/ml in supplemented culture medium (RPMI 1640 with 25 mM HEPES, 10% fetal bovine serum, 2 mM L-glutamine, 1% penicillin-streptomycin, 0.2 mM 2-mercaptoethanol). Plates were incubated at 37°C with 5% CO2 for 72 h. The cell supernatant was removed from each well, centrifuged for 5 min at 5,000 rpm, transferred into sterile tubes, and stored at –80°C. IFN- concentrations were assayed by enzyme-linked immunosorbent assay (ELISA) by diluting samples to 1:10 and using the manufacturer's protocols for the OptEIA IFN- kit (BD Pharmingen, San Diego, CA).
Anti-P. chabaudi ELISA. Lysate of P. chabaudi antigen was used as a capture antigen, and the protein concentration was estimated by using a bicinchoninic acid protein assay (Pierce, Rockford, IL). Microtiter plates were coated overnight at 4°C in carbonate buffer with 5 μg of P. chabaudi antigen/ml for immunoglobulin G (IgG) or 20 μg 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) for IgG 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 and 1:50 for IgG1 and IgG2c in PBS (sera dilutions were based on optimization assays). 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 PBS for 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 optical density was measured at 450 nm, and the samples were expressed as a percentage of the positive control run on the same microtiter plate.
Microarrays. Viable WBCs were adjusted to 5 x 106 cells/ml by dilution with PBS and stored in liquid nitrogen. For RNA extraction, TRIzol LS (Invitrogen, Carlsbad, CA) was added to cells, followed by high-speed disruption in a FastPrep 120 Instrument (Q-Biogene, Irvine, CA) at speed 5 for 15 s. Homogenates were subsequently processed according to the manufacturer's protocol (Invitrogen). RNA pellets were resuspended in nuclease-free water. The quantitation of RNA was performed by using a Beckman DU640 spectrophotometer, and an RNA quality assessment was determined by RNA Nano LabChip analysis on an Agilent Bioanalyzer 2100. Equivalent aliquots (2.5 μg) of RNA from three animals/treatment group were pooled, and three separate pools were processed on separate Affymetrix GeneChips (n = 3 GeneChips/time point/treatment group) (16). The variability among the three GeneChips, within each treatment group at each time point ranged from 0.83 to 2.25%.
The processing of RNA for GeneChip analysis was in accordance with the methods described in the Affymetrix GeneChip Expression Analysis Technical Manual, revision three, as previously reported (14). Hybridization cocktails were prepared as recommended prior to pipetting into the Affymetrix GeneChips (Murine Genome MOE430A). Hybridization and the signal amplification protocol for washing and staining of eukaryotic targets were performed as previously described (14). The arrays were scanned at an emission wavelength of 570 nm at 2.5-mm resolution in the GCS3000 laser scanner (Affymetrix). The intensity of the hybridization for each probe pair was computed by GCOS 1.1 software. For more detailed methods, please refer to the Web site of the Malaria Research Institute Gene Array Core Facility at the Johns Hopkins Bloomberg School of Public Health (http://jhmmi.jhsph.edu/). Primary analysis of microarrays consisted of a quality assessment of the hybridization for each sample. The ratios of signal for probe sets at the 5' and 3' regions of the housekeeping genes were calculated and monitored as an indication of the transcript quality for each sample.
Real-time RT-PCR for microarray validation. Custom primer and probe sets were generated for each target gene by using Primer Express 2.0 software (Applied Biosystems, Foster City, CA). Reverse transcription (RT) of 1.5 μg of RNA from each individual animal sample (n = 8 to 10/time point/treatment group) was conducted with oligo(dT)16 primers and according to the manufacturer's protocol for the SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen). cDNA was amplified in a reaction mixture containing 50 μM concentrations of the primers, a 250 μM concentration of the probe, and the TaqMan Universal PCR mix according to the manufacturer's protocol (Applied Biosystems). All reactions were run in optical 96-well plates using the Prism 7000 Sequence Detection System (Applied Biosystems). Serial dilutions of pooled cDNA from randomly selected animals from each treatment group at each time point 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 were reported relative to the values from same-treatment uninfected control animals.
Statistical analyses. The proportion of animals that survived infection was compared among experimental groups by chi-square analyses. Survival curves were compared between sexes by using Cox Hazard regression analyses and log-rank analyses. Parasitemia, anemia, body temperature, and body mass changes were analyzed by using mixed analyses of variance (ANOVAs), with one within subjects variable (days p.i.) and one between subjects variable (treatment group). Antibody responses and splenomegaly were analyzed by using two-way ANOVAs with two between subjects variable (day p.i. and treatment group). IFN- concentrations were assessed by using a one-way ANOVA. Significant interactions were further analyzed by using planned comparisons or the Tukey method for pairwise multiple comparisons. The combined contribution of parasitemia, anemia, weight loss, and hypothermia to mortality was investigated by using multiple linear regression. Correlations among dependent variables were assessed by using Pearson product moment correlational analyses. Mean differences were considered statistically significant if P was <0.05.
For microarray analyses, data were preprocessed by using GC-RMA in GeneSpring 7.2 (13). Gene expression patterns for each gene from infected animals were normalized to the expression levels from same-treatment uninfected control (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 compare gene expression patterns between intact males and females and between gdx and intact animals of the same sex, and mean differences were considered statistically significant if P was <0.05. Because thousands of statistical tests were performed on 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 that for the genes that had a statistically significant difference, the false discovery rates 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 the NCBI Gene Expression Omnibus (GEO accession GSE4324; www.ncbi.nlm.nih.gov/geo/).
RESULTS
Intact males are more likely to die from infection and recover more slowly from infection-induced weight loss and anemia than intact females. To determine whether responses to blood-stage malaria differed between the sexes and were affected by the presence or absence of gonadal steroids, intact and gdx C57BL/6 mice were inoculated with P. chabaudi AS-infected erythrocytes, and body mass, anemia, parasitemia, antibody responses, IFN- concentrations, and expression levels of immune-related genes were monitored. Significantly more intact C57BL/6 males (7 of 25) died from P. chabaudi infection than gdx males (0 of 20), intact females (1 of 24), and gdx females (1 of 21) (P = 0.005; Fig. 1A). Death from P. chabaudi infection occurred between days 8 and 10 p.i. Levels of parasitemia peaked 7 days after inoculation with P. chabaudi for all animals (P < 0.0001; Fig. 1B). The peak parasitemia was marginally higher for gdx males than for intact males, intact females, 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 infected animals relative to the responses from uninfected same sex and treatment controls. After inoculation with P. chabaudi, body mass decreased significantly through day 10 p.i. and returned to baseline by day 21 p.i. for all animals (P < 0.0001; Fig. 1C). The body mass of intact females, however, returned to baseline faster than for intact males and gdx females, as noted by the rapid weight gain among intact females 14 and 21 days p.i. (P < 0.05; Fig. 1C). After inoculation with P. chabaudi, the percentage 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 from anemia faster than intact males and gdx females, as indicated by the rapid replacement of RBC in intact females by 14 days p.i. (P = 0.06; Fig. 1D). Over the course of infection, the spleen mass increased, whereas the WBC counts decreased, for all animals by 14 and 21 days p.i. (P < 0.001 in each case; data not shown). There were no sex differences or effects of gonadectomy 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 males and females and is altered by gonadectomy. Expression microarrays and real-time RT-PCR were used to measure changes 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 genes arrayed on the mouse MOE430A GeneChip (Affymetrix), 492 genes showed differential expression levels between intact males and intact females, 45 genes were differentially expressed between intact males and gdx males, and 3 genes differed between intact females and gdx females. Because baseline differences in gene expression were observed, gene expression patterns for each gene from infected animals were normalized to the expression levels from same-treatment uninfected control (i.e., day 0) animals (i.e., per gene normalization) (19), which enabled sex or treatment differences in the expression levels of genes 3 to 14 days p.i. to be attributed to infection.
During infection (days 3 to 14 p.i.), 5,182 genes showed significant differences in the relative expression intensity between the sexes, 4,360 genes differed between intact males and gdx males, and 7,013 genes differed between intact females and gdx females (P < 0.05 in each case). Using GO Biological Processes descriptions, we identified 74 unique genes with associated immune function that had expression levels that differed significantly between intact males and intact females during infection (P < 0.05; Fig. 2A and Table S1 in the supplemental material); 53 unique immune-related genes that differed significantly between intact males and gdx males during infection (P < 0.05; Fig. 2B and Table S2 in the supplemental material), and 60 unique immune-related genes that differed significantly between intact females and gdx females during P. chabaudi infection (P < 0.05; Fig. 2C and Table S3 in the supplemental material). Differential expression of genes during P. chabaudi infection was observed 3, 7, and 14 days p.i. (Tables S1 to S3 in the supplemental material); differences in gene expression levels among the groups of mice, however, were most pronounced during peak parasitemia (i.e., day 7 p.i.; Fig. 2).
Among intact mice, males and females exhibited sexually dimorphic expression of several genes associated with innate immunity, antigen presentation, cell adhesion, cytokines, iron metabolism, apoptosis, cellular proliferation, complement, and antibody production during P. chabaudi infection (P < 0.05; Fig. 2A and Table S1 in the supplemental material). Of specific interest, genes involved in the production of IFN- and the development of Th1 immunity were consistently upregulated among intact female mice during peak parasitemia. For example, CCL3 (i.e., MIP-1), CXCL10 (i.e., IP-10), CCR5, IFN-, growth arrest- and DNA-damage-inducible 45 (Gadd45), IL-12R2, and IL-15R, as well as IFN//-inducible genes (e.g., IFN--inducible protein 16 [Ifi16] and IFN-induced protein with tetratricopeptide repeats 2 [Ifit2]) had significantly higher expression levels in intact females than intact males during peak parasitemia (Fig. 2A and Table S1 in the supplemental material). In particular, the expression of IFN- was sixfold higher in intact females than in intact males during peak parasitemia (Fig. 3C). In addition, the expression of regulatory T-cell genes, including IL-10 and TNFR superfamily member 4 (Tnfrsf4; OX-40), was twofold higher among intact female than intact male mice during peak parasitemia (Fig. 4A and B). IFN- inhibits iron metabolism in WBCs (4) and several genes associated with iron regulation, including lactotransferrin (Ltf) and transferrin receptor 2 (TrfR2), were significantly downregulated among intact females compared to intact males (P < 0.05; Fig. 5A and B.
During infection, gonadectomy of male and female mice significantly altered the expression of immune-related genes relative to their intact counterparts. Gonadectomy of male and female mice significantly altered the expression of genes associated with innate immunity, chemokines, cytokines, antigen presention, regulatory responses, cellular proliferation, apoptosis, and signal transduction compared to their intact counterparts (P < 0.05; Fig. 2B and C and Tables S2 and S3 in the supplemental material). Gonadectomy of male mice increased the expression of IL-15R, Gadd45, IFN-induced transmembrane protein 3-like (Ifitm3l), IFN--inducible Puma-g (Gpr109b), Myd88, CCL3, IFN-related developmental regulator 1 (Ifrd1), and TGF- induced (Tgfbi) compared to intact males (Fig. 2B and Table S2 in the supplemental material). Gonadectomy of female mice significantly suppressed the expression of several genes associated with Th1 immunity and regulatory T-cell responses, including Ifrd1, TGF--inducible early growth response 1 (Tieg1) CCR5, IFN-, Gadd45, IL-12R2, and IL-15R, during infection (Fig. 2C and Table S3 in the supplemental material).
Because of the potential problem of false positives associated with microarray analyses, several genes of interest were validated by using real-time RT-PCR. Real-time RT-PCR also was used to further examine gene expression profiles at 21 days p.i. The expression patterns of IL-12R2, IFN-, Gadd45, IL-10, Tnfrsf4, Ltf, TrfR, and -actin in WBCs from individual animals were examined, and the expression levels of each gene from infected animals were analyzed relative to the expression levels of each gene from uninfected same-treatment control animals. The expression of the housekeeping gene -actin was similar among intact and gdx males and females days 0 to 21 p.i. (P > 0.05). Consistent with our microarray results, the expression of genes associated with the synthesis of IFN-, including IL-12R, Gadd45, and IFN-, was higher among intact females than intact males, gdx males, and gdx females 7 days p.i. (P < 0.01 in each case; Fig. 3). Although microarray analyses revealed that gdx males had a higher expression of 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 isolated from intact females produced more IFN- than did WBCs isolated 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 highest expression 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 females had higher expression of regulatory genes, including IL-10 and Tnfrsf4, than intact males during peak parasitemia (P < 0.001 in each case; Fig. 4); real-time RT-PCR results further indicated that gonadectomy of females significantly suppressed the expression of IL-10 and Tnfrsf4 and gonadectomy of males enhanced the expression of IL-10 at day 7 p.i. (Fig. 4C and D). Microarray and real-time RT-PCR analyses consistently illustrated that the expression of genes associated with iron homeostasis, including Ltf and TrfR, was higher among intact males than among intact females 14 days p.i. and that gonadectomy of male mice significantly reduced the expression of the TrfR (P < 0.001 in each case; Fig. 5).
During the recovery phase of infection, intact females have higher antibody responses than either intact males or gdx females. To eliminate the erythrocytic stage of Plasmodium parasites, both humoral and cell-mediated effector mechanisms are required. Anti-P. chabaudi IgG increased over time for all animals and peaked at 14 to 21 days p.i. (P < 0.001; Fig. 6A). Anti-P. chabaudi IgG antibody responses, however, were higher among intact females than among intact males at 14 to 21 days p.i; gonadectomy of female mice reversed this sex difference by 21 days p.i. (P < 0.01; Fig. 6A).
To assess whether different subsets of helper T cells were activated by males and females in response to infection, subclasses of IgG were examined. Anti-P. chabaudi IgG1 responses increased over the course of infection and were highest at 14 to 21 days p.i. for all animals (P < 0.001; Fig. 6B). Anti-P. chabaudi IgG1 responses were significantly higher among intact females than intact males at 14 days p.i. (P < 0.01; Fig. 6B). C57BL/6 mice do not possess the gene for IgG2a but express a novel isotype, IgG2c, that is indicative of a Th1-mediated response (28). Anti-P. chabaudi IgG2c responses peaked 14 to 21 days p.i. for all animals (P < 0.001; Fig. 6C) but did not differ significantly among the experimental groups of mice (P > 0.05; Fig. 6C).
Utilization of immunodeficient mice reveals that innate immunity may mediate sexually dimorphic responses to P. chabaudi infection. To determine the roles of innate and acquired immune responses, as well as the specific effects of IFN-, as mediators of sex differences in P. chabaudi infection, we inoculated adult WT, TCR–/–, μMT, RAG1, and IFN-–/– mice with P. chabaudi parasites and monitored sex differences in parasitemia, body mass, rectal temperature, anemia, and mortality. Peak parasitemia occurred for all mice at 7 days p.i. and was followed by a period of reduced parasitemia in WT, RAG1, and μMT mice (Fig. 7). In contrast, among TCR–/– 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, μMT, and IFN-–/– mice compared to their female counterparts (P < 0.05 in each case; Fig. 7). Among TCR–/– mice, although peak parasitemia was similar between males and females, males had a higher proportion of parasitized erythrocytes at 10 days p.i. (because only one TCR–/– male was still alive days 14 to 21 p.i, statistical comparisons could not be made at these time points) (P < 0.05; Fig. 7). Peak parasitemia was similarly high for both male and female RAG1 mice (P > 0.05; Fig. 7).
A Cox Hazard regression analysis across all strains of mice revealed that males were 3.5 times (95% confidence interval of 2.13 to 5.71) more likely to die from blood-stage malaria infection than were females. Based on log-rank analyses, survival was significantly prolonged for female compared to male RAG1, TCR–/–, and μMT mice (P < 0.001 in each case; Fig. 7). As a result, the average day of death was significantly earlier for male than female RAG1 (P < 0.01 [males, 8 days p.i.; females, 14 days p.i.]), TCR–/– (P < 0.001 [males, 10 days p.i.; females, 21 days p.i.]), and μMT (P < 0.01 [males, 8 days 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 male IFN-–/– mice began dying significantly sooner after inoculation with P. chabaudi than did females (P < 0.05; Fig. 7). Among male mice, deletion of T cells, B cells, or both lymphocyte populations significantly increased the likelihood of dying from blood-stage malaria infection compared to WT mice (P < 0.001 in each case). Among female mice, the absence of IFN-, B cells, or both T and B cells significantly increased mortality from P. chabaudi infection compared to WT 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 had less severe hypothermia during infection and generally returned to baseline faster than males (P < 0.05 in each case; Fig. 8) . Among RAG1 and TCR–/– mice, males lost more weight than females by 3 to 7 days p.i. (P > 0.05; Fig. 8). Female RAG1, μMT, and TCR–/– mice consistently developed less severe hypothermia than their male counterparts (P < 0.01 in each case; Fig. 8). Male and female RAG1, μMT, and TCR–/– mice exhibited a similar decrease in the percentage of RBCs through day 7 p.i. (P > 0.05 in each case; Fig. 8). Among IFN-–/– mice, females exhibited an initial decrease in body mass, the percentage of RBCs, and body temperature that was followed by an early return to baseline by day 10 p.i. and a subsequent relapse 14 days p.i., resulting in a pattern that was significantly different from that of the WT female mice (P < 0.01 in each case). Conversely, male IFN-–/– mice demonstrated a more consistent decline in body mass, percentage of RBC, and body temperature through day 10 p.i., followed by a return to baseline by day 14 p.i., which was not significantly different from WT males (P > 0.05 in each case). The dimorphic response of IFN-–/– mice to infection resulted in IFN-–/– males having more severe weight loss, anemia, and hypothermia than IFN-–/– females at 10 days p.i. and IFN-–/– females exhibiting increased morbidity 14 days p.i. (P < 0.001 in each case; Fig. 8).
Multiple linear regression analyses revealed that at 7 days p.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 hypothermia, but not parasitemia, anemia, or weight loss, in both males (P < 0.001) and females (P < 0.001), regardless of their immunodeficiency.
DISCUSSION
In the present series of studies, gonadally intact WT males were more likely than intact WT females to die after P. chabaudi infection, exhibit slower recovery from infection-associated weight loss, hypothermia, and anemia, have reduced IFN--associated gene expression and IFN- production during peak parasitemia, and produce less antibody during the recovery phase of infection. Gonadectomy of male and female WT mice altered these sex-associated differences, suggesting that sex steroid hormone, in particular androgens and estrogens, may modulate immune responses to infection.
Infection of C57BL/6 mice with P. chabaudi causes transient anemia, weight loss, and hypothermia. These sequelae, combined with elevated parasitemia, are associated with increased mortality from blood-stage malaria infection (42). In the present study, elevated parasitemia at 7 days p.i. and severe hypothermia at 10 days p.i. were significant predictors of death from P. chabaudi in both male and female mice. Susceptibility to P. chabaudi also is associated with low inflammatory responses during the acute phase of infection (46). We observed that individuals with the highest concentrations of IFN- tended to have the lowest levels of parasitemia, which may contribute to the increased likelihood of surviving infection among females compared to males.
Characterization of the immunological causes of sex differences in Plasmodium infection using immunodeficient mice revealed that sex differences in parasitemia, morbidity (as measured by changes body mass, RBC loss, and development of hypothermia), and mortality were still apparent in the absence of T cells, B cells, or both cell populations. Resistance to blood-stage malaria was compromised in immunodeficient male and female mice relative to their WT counterparts, since both male and female mice deficient in T cells, B cells, or both cell populations experienced chronic parasitemia and increased mortality compared to WT mice. Thus, acquired immunity plays a critical role in mediating the clearance of parasites and recovery from infection. These immunodeficiencies, however, were consistently more detrimental to males than to females because the morbidity and mortality rates, as well as the levels of parasitemia, were still higher in RAG1, TCR–/–, and μMT male mice than in female mice. Our data suggest an important role of innate immunity, including the activity of macrophages, dendritic cells, and NK cells (all of which are functional in RAG1, TCR–/–, and μMT mice), in mediating the sexual dimorphism in susceptibility to P. chabaudi.
To further assess immunological differences in response to malaria infection, microarrays and real-time RT-PCR were used to map the expression levels of immune-related genes in WBCs over the course of P. chabaudi infection. Previous studies illustrate that the administration of pharmacological doses of testosterone suppresses the expression of immune-related genes, including Gadd45 and CXCL10, in the livers, but has no effect on gene expression in the spleens, of female mice infected with P. chabaudi and tested at day 8 p.i. only (22). Female mice injected with high doses of testosterone also exhibit reduced antibody production and expression of major histocompatibility complex class II on spleen cells but do not show altered cytokine production in the spleen during the acute phase of infection compared to intact female mice injected with vehicle (2). Whether immunological parameters differ between males and females during infection has not been systematically examined over the course of infection prior to the present study. We demonstrate that females respond to P. chabaudi infection with a heightened IFN--mediated response during the acute phase of infection, an elevated antibody response during the chronic phase of infection, and reduced manifestation of disease compared to males. We also demonstrate that gonadectomy of females reverses the female-typic immune response to infection, suggesting that estradiol may play a more central role than previously assumed. As a result, our data demonstrate that, in addition to the role played by testosterone, estrogens may play a pivotal role in immune modulation of malaria infection.
The timing of Th1 and Th2 responses is critical for protection against many Plasmodium parasites (26, 46). Elevated Th1-mediated responses during the acute phase of infection are required to reduce peak parasitemia, and activation of Th2-mediated responses during the chronic phase of infection is required to clear parasites (46). Macrophage and dendritic cell-derived IL-12 is important for the development of Th1 phenotypic responses, such as the production of IFN-. Mice that are resistant to blood-stage Plasmodium infection produce more splenic IL-12 and IFN- prior to and during peak parasitemia than do susceptible mice during P. chabaudi infection (41, 48, 49). In the present study, intact females exhibited higher expression of IFN--associated genes, including IL-12R2 and IFN-, and produced more IFN- than did intact males during peak parasitemia. Genes involved in the synthesis of IFN-, including Gadd45, which induces phosphorylation of STAT4 by the p38 pathway (33), showed a similar pattern of expression. IL-15 promotes Th-1-dependent immunity against P. chabaudi infection (12), and the expression of IL-15R was higher on WBCs from intact females than from intact males and was reversed by gonadectomy of both sexes during peak parasitemia. Several chemokines, including CCL3 and CXCL10, that preferentially attract Th1 cells (40), were upregulated in the WBCs from intact females. The chemokine receptor CCR5, which binds CCL3, is expressed at high levels on Th1 cells (27), and is regulated by estrogens (31), also was upregulated on WBCs from intact females. The removal of estrogens via gonadectomy significantly reduced the expression of these Th1-related genes and IFN- production in a manner similar to the reported effects of estradiol on IFN- responses against Leishmania mexicana (44).
If IFN- is the primary mediator of sex differences in response to P. chabaudi, then elimination of IFN- should reduce the sexual dimorphism. Although IFN-–/– males had higher peak parasitemia than did conspecific females, similar proportions of male and female IFN-–/– mice died after infection, providing further evidence that sex differences during blood-stage malaria infection may be influenced by the production of IFN-. The removal of IFN- was consistently more detrimental to females than to males, as illustrated by the increased mortality rates and relapse in body mass loss, RBC loss, and hypothermia in female IFN-–/– compared to female WT mice. It should be noted, however, that our observation of a similar rate of survival among male and female IFN-–/– mice after P. chabaudi AS inoculation is in contrast to previous data 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 mediator of sex differences in response to blood-stage malaria infection.
After peak parasitemia, Th1 responses begin to decline and Th2 phenotypic responses increase in mice that are resistant to Plasmodium infection (46). Intact females had significantly higher anti-P. chabaudi IgG1 responses than males 14 days after infection, suggesting that Th2 responses may be elevated among intact females during the recovery phase of infection. Among intact females, elevated IFN- during the acute phase of infection may induce increased antibody production during later phases of malaria infection (49). Overall, intact females developed higher antibody responses than intact males, and removal of the ovaries and, hence, the primary source of estrogens, significantly suppressed antibody production. Consequently, estrogens potentiate (10) and the administration of testosterone to female mice reduces (2) antibody production by B cells. Although intact females produced higher antibody responses against P. chabaudi, immunoglobulin gene transcripts were elevated in WBCs from intact males, suggesting that there are posttranscriptional differences between the sexes. Although antibody production was suppressed in intact WT males, the removal of B cells in μMT mice did not abolish the sexual dimorphism in response to P. chabaudi infection, suggesting that B cells may not be critical mediators of sex differences in malaria infection.
Regulatory responses, including the production of TGF- and IL-10, mediate the timing and shift from Th1 to Th2 responses and are critical for mitigating pathology caused by prolonged elevation of inflammatory responses during the course of malaria infection (25, 35). Although sex differences in TGF- expression were not observed, IL-10 expression was higher in WBCs from intact females than from intact males, and gonadectomy of females reduced, whereas gonadectomy of males increased, the transcription of IL-10. Whether the differential expression of IL-10 is mediated by macrophages, T cells, or both requires additional investigation. The expression of several genes associated with activated CD4+ CD25+ T cells (29), including Tnfrsf4, Ltb, CCL3, and Gadd45, was higher among intact females than among intact males and was reduced by gonadectomy of female mice in the present study. Estrogens can augment the expansion of CD4+ CD25+ T cells in mice (38). Previous data illustrate that administration of high doses of testosterone to intact females has no effect on production of IFN- or IL-10 (2); thus, sex differences in cytokine and possibly chemokine production during malaria infection are likely mediated by estrogens and not androgens, as previously hypothesized.
In addition to being a key player in Th1 immunity against malaria, IFN- plays a critical role in iron metabolism in WBCs. IFN- reduces iron uptake by macrophages both indirectly through nitric oxide and iron-regulatory proteins and directly by causing downregulation of iron receptors, such as the TrfR (4). In the present study, the transcription of TrfR was downregulated among intact females and gdx males compared to intact males. Elevated IFN- production among intact females may be one mechanism mediating reduced expression of the TrfR. Whether IFN- alters TrfR expression directly or through NO-mediated degradation of iron regulatory proteins requires additional investigation. In the present study, inducible NO synthase expression did not differ between the sexes, suggesting that the direct effects of IFN- on TrfR expression may be involved. Although iron supplementation is beneficial for resolving anemia, iron chelation, which may be regulated by IFN-, effectively reduces the incidence and severity of P. falciparum infection in children (9, 36). In mice, iron-supplemented diets increase parasitemia and mortality following P. chabaudi infection to a greater extent among males than females (11), suggesting that dimorphic iron regulation may contribute to differential responses to infection.
Although sex differences in response to malaria infection are reported among both adults and children, little is known about the mechanisms mediating these differences or whether these sex differences will affect responses to drug treatments or vaccines. Men and women differ in disease manifestations after infection; men are more likely to develop lymphomas, whereas women are more likely to develop anemia, after infection (5, 6, 34). In general, most studies of malaria in human populations do not distinguish between the responses of males and females and, thus, the prevalence of sex differences may be underreported (1). A few studies do, however, clearly illustrate that men are more susceptible than women. For example, several studies indicate that men tend to have higher parasitemia than women (23, 32, 52). In a recent prospective study of imported malaria cases in France, men reported more severe symptoms of malaria infection (e.g., chills, fever, and low platelet counts) than did women (7). Among Ghanaian school children, although the prevalence of P. falciparum infection does not differ between the sexes, the parasite density is significantly higher for boys than girls around puberty (i.e., from ages 8 to 16), suggesting that circulating sex steroids may influence this outcome (23). Although significantly more men die from infectious diseases than women (37), sex differences in mortality from Plasmodium infection are not consistently reported. There are contradictory findings from a study in India, wherein mortality rates were reportedly higher in women than men after infection with P. falciparum (20).
Our studies with a rodent malaria model indicate that males and females respond differently to malaria infection. Reduced rates of parasitemia and faster recovery from hypothermia likely contribute to reduced death rates among females compared to males. The immunological mechanisms that underlie the dimorphism in morbidity and mortality likely involve the heightened IFN- and regulatory T-cell response observed in female mice. These data make a compelling case for the reevaluation of previous epidemiological studies as well as development of prospective studies that are designed to systematically test the hypothesis that males and females differ in their responses to malaria infection. Although sex differences in mortality and parasitemia following Plasmodium infection are reported among rodents, the immunological mechanisms mediating these differences have not been adequately examined. The data from the present study provide candidate immunological pathways that differ between the sexes and that are significantly altered by the absence of estrogens during blood-stage malaria infection.
ACKNOWLEDGMENTS
We thank Mary Stevenson and Mifong Tam for providing the initial stock of P. chabaudi parasites and for assistance with assay development. We also thank Marie Diener-West and Gregory Glass for statistical advice and Judith Easterbrook, Autumn Girouard, Michele Hannah, Peter Klein, and Brandon Zonker for technical assistance.
Financial support for this study was provided by The Johns Hopkins Malaria Research Institute.
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.
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