Leukotrienes (LTs) are potent lipid mediators involved in the control of host defense. LTB4 induces leukocyte accumulation, enhances phagocytosis and bacterial clearance, and increases NO synthesis. LTB4 is also important in early effector T cell recruitment that is mediated by LTB4 receptor 1, the high-affinity receptor for LTB4. The aims of this study were to evaluate whether LTs are involved in the secondary immune response to vaccination in a murine model of Histoplasma capsulatum infection. Our results demonstrate that protection of wild-type mice immunized with cell-free Ags from H. capsulatum against histoplasmosis was associated with increased LTB4 and IFN-γ production as well as recruitment of memory T cells into the lungs. In contrast, cell-free Ag-immunized mice lacking 5-lipoxygenase−/−, a critical enzyme involved in LT synthesis, displayed a marked decrease on recruitment of memory T cells to the lungs associated with increased synthesis of TGF-β as well as IL-10. Strikingly, these effects were associated with increased mortality to 5-lipoxygenase−/−-infected mice. These data establish an important immunomodulatory role of LTs, in both the primary and secondary immune responses to histoplasmosis.

Infection with the dimorphic fungus Histoplasma capsulatum ranges from a mild asymptomatic illness to a progressive and disseminated disease. The effective immune response to H. capsulatum relies on adaptive immunity and the collaboration between infected macrophages with CD4+ and CD8+ T cells (1, 2). This interaction results in increased amounts of Th1 and Th1-related cytokines, including IL-12, IFN-γ, TNF-α, and GM-CSF, which are critically involved in the protective immune response in H. capsulatum-infected mice (3, 4, 5, 6, 7). In opposition is the anti-inflammatory cytokine TGF-β, which counterregulates Th1 cell differentiation. TGF-β blockade of Th1 cell differentiation is associated with reduced IL-12Rβ2, T-bet, and STAT4 expression, whereas T-bet expression inhibits IFN-γ production during recall responses (8, 9, 10).

In addition to cytokines, another prominent class of molecules involved in the control of host defense are leukotrienes (LTs),3 lipid mediators derived from arachidonic acid (AA) metabolism. LTs are derived from the metabolism of AA through the enzyme 5-lipoxygenase (5-LO), in concert with its helper protein, 5-LO-activating protein (FLAP). The 5-LO oxygenates AA to the intermediate 5-hydroperoxyeicosatetraenoic acid, which is either enzymatically reduced by 5-LO to the unstable epoxide LTA4 or alternatively is reduced to 5-hydroxyeicosatetraenoic acid. LTA4 can be hydrolyzed to form LTB4 or can be conjugated with glutathione to form the cysteinyl LTs, LTC4, LTD4, and LTE4 (11). The role of endogenous LTs in host defense during microbial infection was first demonstrated by Bailie et al. (12), who reported that 5-LO deficiency impaired survival and pulmonary bacterial clearance in a model of Klebsiella pneumoniae pneumonia. Subsequent studies have documented a protective function of endogenous LTs in several experimental models, including bacterial peritonitis (13), tuberculosis (14), and parasitic infection (15, 16). We have recently demonstrated an important role for LTs in the primary immune response to H. capsulatum (17). Reduction in LT synthesis used by administration of MK886, an inhibitor of the FLAP, resulted in 100% mortality in sublethally H. capsulatum-infected mice, higher numbers of lung and spleen CFU, and impaired NO production.

The effector functions involved in innate immune responses that are influenced by LTs include direct effects on leukocyte accumulation (18, 19, 20, 21), microbial phagocytosis and killing (15, 22, 23, 24), and indirect effects mediated by elaboration of other inflammatory molecules (15, 17, 24). For example, the effects of LTs on the secretion of cytokines have been reported in vitro and in vivo. We demonstrated that inhibition of LT synthesis in animals infected with H. capsulatum resulted in lower IL-2, IL-5, IL-12, and IFN-γ levels (17). Moreover, in vitro, pharmacological blockade of LTB4 receptor 1 (BLT1) resulted in increased IL-10 production (25). DiMeo et al. (26) demonstrated that LPS-stimulated spleen cells from 5-LO-deficient mice produced less IL-12p70, IL-6, TNF-α, and IFN-γ. LTB4 has also been shown to exhibit potent chemotatic activity for in vitro activated effector CD4+ and CD8+ lymphocytes, which is mediated by BLT1, the high-affinity receptor for LTB4 (27, 28).

Because LTs enhance both phagocyte function and T cell function, we hypothesized that LTB4 might be an important mediator involved in the protective response to vaccination designed in a murine model of H. capsulatum infection. By using a standardized preparation of extracts containing H. capsulatum cell-free Ags (CFAgs), we observed that immunization was able to protect mice against a lethal inoculum of H. capsulatum and significantly reduce fungal burden in the lungs and spleens of infected mice (29). In the present study, we demonstrate that LTs play a key role in the secondary immune response induced by CFAg immunization through modulation of cytokine synthesis and generation or migration of memory T cells against H. capsulatum.

Six- to 8-wk-old 5-LO−/− (129-Alox5tm1Fun) and strain-matched wild type (WT) were obtained from Jackson ImmunoResearch Laboratories, and male C57BL/6 mice were bred in the Faculdade de Ciências Farmacêuticas de Ribeirão Preto (Universidade de São Paulo, Brazil). All experiments were approved and conducted in accordance with the guidelines of the Animal Care Committee of the University. Infected animals were kept in biohazard facilities and housed in cages within a laminar flow safety enclosure under standard conditions.

The clinical isolate of H. capsulatum obtained from a patient at the Hospital das Clínicas of Faculdade de Medicina de Ribeirão Preto (Universidade de São Paulo, Brazil) was grown on Sabouraud-dextrose-agar at room temperature for 2 mo, and conidia morphology was observed. Later, mycelia were grown in brain-heart infusion (BHI)-agar-blood at 37°C for 15 days to convert them into the yeast form, and morphology was also confirmed. After this characterization, preparation of H. capsulatum yeast cells was performed, as previously described (17). Yeast cells were used when fluorescein diacetate and ethidium bromide staining (30) revealed their viability to be ≥90%.

Lethal challenges of 106 yeasts intratracheal (i.t.) in C57BL/6 mice and 3 × 106 yeasts in 5-LO−/− (129-Alox5tm1Fun) and strain-matched WT mice were used to evaluate mortality rates and cellular immunity in subsequent experiments. The LD50 for both strains were previously determined (data not shown).

CFAgs were produced according to Camargo et al. (31), with minor modifications (29). Briefly, ∼300 mg of H. capsulatum yeasts growth on BHI-agar-blood slants were collected and suspended in 1 ml of PBS, mixed on a vortex-mixer, and centrifuged at 10,000 × g for 60 s. The supernatant fluid was collected, and the procedure was repeated. This material was filtered, and its protein content was estimated using Coomassie Protein Assay Reagent Kit (Pierce), using albumin standard curves prepared according to the manufacturer’s instructions.

CFAg preparation was emulsified in CFA (v/v) at a final concentration of 200 μg/ml. Mice were injected s.c. with 50 μg/0.25 ml emulsified CFAgs. One week later, mice received the same amount of the preparation emulsified in IFA. After 2 wk, mice received an i.v. booster of 5 μg of CFAg preparation in 0.1 ml. The control group received an equal amount of BSA suspended in the same adjuvants during the immunizations and a booster of 5 μg of BSA in 0.1 ml.

C57BL/6 mice were divided into two groups. Group 1 animals were treated with MK 886 perorally (p.o.) by gavage (5 mg/kg/0.5 ml) 1 h before immunization s.c. with CFAgs, and again every 24 h until day 21 (1 day before infection with H. capsulatum). This group is denominated CFAgs + MK886Immunization. Group 2 animals were treated with MK 886 p.o. by gavage (5 mg/kg/0.5 ml) 1 h before rechallenge with H. capsulatum, and again every 24 h until day 60. This group is denominated CFAgs + MK886Postinfection. Control animals were inoculated i.t. with PBS and were given water p.o. for 60 days.

For cytokine and LTB4 measurement, lungs were removed on day 7 postinfection. Tissues were homogenized (Mixer Homogenizer; Labortechnik) in 2 ml of RPMI 1640, centrifuged at 1500 × g, filtered, sterilized, and stored at −70°C until assayed. Quantification of LTB4 in the samples was performed by specific enzyme immunoassay (Amersham Biosciences) following the supplier’s instructions. The OD of samples was determined at 412 nm in a microplate reader (μ Quant Bio; TEK Instruments), and concentrations of eicosanoids were calculated based on a standard curve. The sensitivity for LTB4 was <4 pg/ml.

Commercially available ELISA Abs were used to measure TNF-α, IL-1α, IL-4, IL-10, IL-12 (p70), active TGF-β1, and IFN-γ (BD OptEIA ELISA sets; BD Pharmingen). Keratinocyte-derived chemokine (KC) was measured by sandwich ELISA (R&D Systems). The cross-reactivity with murine TGF-β was >90%. The sensitivity for cytokines was >30 pg/ml. Reactions were performed on 96-well ELISA plates (Ultra-High Binding EIA Plates; Corning Glass), and the ODs were read on a microplate reader (μ Quant Bio; TEK Instruments) at a wavelength of 450 nm. The cytokine concentration in each sample was estimated by interpolation of sample ODs with the cytokine standard by a four-parameter curve-fitting program.

NO production was assessed by measuring the amount of nitrite (NO2) in lung homogenates, obtained as described above, using the Griess reaction, as previously described (17). Values were determined using a standard curve with serial dilutions of NaNO2 (Sigma-Aldrich).

Recovery of H. capsulatum was performed, as previously described by Sá-Nunes et al. (32). Two hundred-microliter samples from each suspension were collected, and serial dilutions (10-fold) were plated on BHI-agar-blood slants. After incubation at 37°C for 21 days, the burden of H. capsulatum was assessed. Results are expressed as CFU/g lung or CFU/spleen.

Lung leukocytes were adjusted to a concentration of 1 × 105 cells/0.1 ml and washed with staining buffer (consisting of PBS (pH 7.4), 2% BSA, and 0.02% sodium azide). FcRs were blocked by the addition of unlabeled anti-CD16/32. The proportion of cells bearing a memory phenotype was determined by staining with appropriate concentrations of fluorochrome-conjugated Abs specific for murine CD4+CD44highCD62L and CD8+CD44highCD62L (BD Pharmingen). The samples were washed and fixed in 2% paraformaldehyde. The results were calculated by percentage of CD4+ or CD8+ T cells with memory phenotype (CD4+CD44highCD62Llow/neg or CD8+CD44highCD62Llow/neg).

The log-rank test was used to assess differences in survival. ANOVA, with Bonferroni’s multiple comparison as a post test, was used to analyze differences in delayed-type hypersensitivity (DTH) responses, cytokine, LTs, and memory T cells. Value of p < 0.05 was considered statistically significant.

To determine whether LTs are required for the generation of the protective immune response, 5-LO-deficient mice were immunized with CFAgs and challenged with a lethal inoculum of H. capsulatum. All WT and 5-LO−/− mice that had been immunized with BSA (irrelevant Ag) succumbed to infection by 17 days postinfection (Fig. 1 A). However, 5-LO−/− BSA-immunized mice succumbed to infection 5 days before WT-BSA. Similarly, only 17% of the 5-LO−/−-CFAg mice survived until 30-day postinfection. Conversely, 83% of WT CFAgs survived until the end of the observation period.

FIGURE 1.

Effect of endogenous LT synthesis inhibition on mouse survival rate and on fungal recovery. A, Survival of CFAg- and BSA-immunized mice rechallenged with lethal dose of H. capsulatum. Mice were monitored for 30 days (n = 5–6). Fungal burden in lungs (B) and spleen (C). Tissue samples were harvested at 7 days of H. capsulatum infection. Data are expressed as mean ± SEM from two experiments. #, p < 0.05 vs WT BSA; ξ, p < 0.05 vs WT CFAgs.

FIGURE 1.

Effect of endogenous LT synthesis inhibition on mouse survival rate and on fungal recovery. A, Survival of CFAg- and BSA-immunized mice rechallenged with lethal dose of H. capsulatum. Mice were monitored for 30 days (n = 5–6). Fungal burden in lungs (B) and spleen (C). Tissue samples were harvested at 7 days of H. capsulatum infection. Data are expressed as mean ± SEM from two experiments. #, p < 0.05 vs WT BSA; ξ, p < 0.05 vs WT CFAgs.

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In another set of experiments, lungs and spleen were harvested from infected animals at day 7 postinfection and examined for fungal burden. We found significant increase in the CFU numbers in WT and 5-LO−/− BSA-immunized mice (p < 0.001 and p < 0.05) when compared with WT CFAg mice (Fig. 1, B and C). Fungal burden in the lungs and spleen of 5-LO−/− CFAg mice was significantly higher from that in WT CFAg mice on day 7 (p< 0.001) (Fig. 1, B and C). Moreover, the number of yeasts recovered in spleen at day 7 was higher in 5-LO−/− BSA-immunized mice when compared with WT BSA- or CFA-immunized mice (Fig. 1 C).

Because the lack of 5-LO abrogated the protective immune response induced by CFAgs, we measured LTB4 in the lung after H. capsulatum infection. LTB4 levels in the lungs from WT BSA mice were ∼2-fold higher than either PBS-injected or 5-LO−/− BSA mice. Moreover, CFAg immunization in WT mice induced a 10-fold increase in LTB4 production when compared with both groups of BSA-immunized mice and infected with H. capsulatum (WT or 5-LO−/− BSA). In contrast, the levels of LTB4 in lungs of 5-LO−/− mice immunized with either CFAgs or BSA were not altered when compared with uninfected mice (Fig. 2 A).

FIGURE 2.

LTB4 and NO2 synthesis in lung tissue. Enzyme immunoassay quantification of LTB4 concentrations in lungs from mice submitted to either i.t. PBS injection (uninfected) or CFAg- and BSA-immunized mice and rechallenged with lethal dose of H. capsulatum. LTB4 (A) and NO2 (B) levels were quantified in the lungs of animals at day 7 of infection, as described in Materials and Methods. Data are presented as mean ± SEM and are representative of one of three independent experiments (n = 6). ∗, p < 0.05 vs uninfected; #, p < 0.05 vs WT BSA; ξ, p < 0.05 vs WT CFAgs.

FIGURE 2.

LTB4 and NO2 synthesis in lung tissue. Enzyme immunoassay quantification of LTB4 concentrations in lungs from mice submitted to either i.t. PBS injection (uninfected) or CFAg- and BSA-immunized mice and rechallenged with lethal dose of H. capsulatum. LTB4 (A) and NO2 (B) levels were quantified in the lungs of animals at day 7 of infection, as described in Materials and Methods. Data are presented as mean ± SEM and are representative of one of three independent experiments (n = 6). ∗, p < 0.05 vs uninfected; #, p < 0.05 vs WT BSA; ξ, p < 0.05 vs WT CFAgs.

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One of the most important microbicidal molecules involved in the control of intracellular pathogens is NO. In addition, it has been shown that LTB4 induces NO secretion and inducible NO synthase expression (15). Our results demonstrated that H. capsulatum infection in WT CFAg mice induced the increase of NO2 production by ∼4-fold when compared with WT and 5-LO−/− BSA mice. However, the deficiency of 5-LO abolished the increased NO2 in the lung induced by CFAg immunization (Fig. 2 B).

Production of Th1-related cytokines is critically important in the host resistance to H. capsulatum (33). Therefore, we examined cytokine production in lung homogenates from BSA-immunized or CFAg-immunized mice after 7 days of infection. H. capsulatum infection in CFAg WT mice induced a 2-fold increase in TNF-α production when compared with BSA-immunized mice (WT or 5-LO−/−-BSA). However, the levels of TNF-α in lungs of 5-LO−/− CFAg mice were not different from those of WT CFAg mice (Fig. 3 A).

FIGURE 3.

Cytokine levels in lung tissue. Mice submitted to either i.t. PBS injection (uninfected) or CFAg- and BSA-immunized mice and rechallenged with lethal dose of H. capsulatum. Lungs removed on day 7 and homogenized in RPMI. TNF-α (A), KC (B), IFN-γ (C), IL-10 (D), and TGF-β (E) levels were determined by ELISA. Data are presented as mean ± SEM of six animals per group and are representative of one of three independent experiments. ∗, p < 0.05 vs uninfected; #, p < 0.05 vs WT BSA; ξ, p < 0.05 vs WT CFAgs.

FIGURE 3.

Cytokine levels in lung tissue. Mice submitted to either i.t. PBS injection (uninfected) or CFAg- and BSA-immunized mice and rechallenged with lethal dose of H. capsulatum. Lungs removed on day 7 and homogenized in RPMI. TNF-α (A), KC (B), IFN-γ (C), IL-10 (D), and TGF-β (E) levels were determined by ELISA. Data are presented as mean ± SEM of six animals per group and are representative of one of three independent experiments. ∗, p < 0.05 vs uninfected; #, p < 0.05 vs WT BSA; ξ, p < 0.05 vs WT CFAgs.

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KC levels in the lungs of infected mice were increased in all groups when compared with those of uninfected animals. However, H. capsulatum infection in CFAg WT mice induced an increase in KC production when compared with BSA-immunized mice (WT or 5-LO−/− BSA). Moreover, the levels of KC in lungs of 5-LO−/− CFAg mice were reduced when compared with WT CFAg mice (Fig. 3 B).

IFN-γ levels were increased only in lung of WT CFAg mice. The deficiency of 5-LO−/− in CFAg-immunized mice impaired the production of IFN-γ in the lungs of these animals. The levels of IFN-γ in lungs of WT BSA, 5-LO−/− BSA, or 5-LO−/− CFAg mice were not different from those uninfected (Fig. 3 C).

The levels of IL-10 were not different between the WT animals immunized with CFAgs or BSA. However, the deficiency of 5-LO enzyme resulted in increased levels of IL-10 when compared with WT CFAgs and WT BSA (Fig. 3 D).

H. capsulatum infection in either WT BSA or 5-LO−/− BSA increased active TGF-β levels above that seen in uninfected mice. CFAg immunization in WT mice impaired the increase of active TGF-β levels when compared with WT BSA. However, the deficiency of 5-LO resulted in the increase of active TGF-β synthesis in the lung of 5-LO−/− CFAg mice when compared with WT CFAg mice (Fig. 3 E).

Levels of IL-1, IL-12, and GM-CSF in lungs of infected WT and 5-LO−/− mice immunized with BSA or CFAgs were similar (data not show). IL-4 in the lung was undetectable 7 days postinfection (data not shown).

The generation of memory cells is an essential component of adaptive immunity and mediates protection in peripheral tissues during a secondary immune response to pathogens. We then investigated whether the increased susceptibility of 5-LO-deficient animals could be attributed to altered proportions of memory cells. The results were calculated by percentage of CD4+ or CD8+ T cells with memory phenotype. WT and 5-LO−/− mice were immunized with CFAgs and BSA and challenged with a lethal dose of H. capsulatum. At day 7 following infection with H. capsulatum, the lungs were analyzed for the presence of memory T cells (Table I and Fig. 4). H. capsulatum infection in WT CFAg mice increased the percentage of pulmonary CD4+CD44highCD62Llow/neg to 41% when compared with WT BSA mice (13.1% CD4+ memory T cells). The deficiency of 5-LO enzyme drastically reduced the percentage of CD4+ memory cells in 5-LO−/− CFAg mice (20% CD4+ memory T cells) and 5-LO−/− BSA mice (19.8% CD4+ memory T cells) infected with H. capsulatum (Fig. 4). Similar results were observed with CD8+CD44highCD62Llow/neg cells (Table I). Thus, these data suggest that LTs are involved in the recruitment or generation of memory T cells.

Table I.

Percentage of memory T cells in the lungs of WT and 5-LO−/− mice immunized with BSA and CFAgs and infected for 7 days with H. capsulatuma

T CellsWT BSA5-LO−/− BSAWT CFAgs5-LO−/− CFAgs
CD4+CD44highCD62Llow/neg 13.8 ± 2.24% 18.29 ± 1.68% 47.50 ± 6.19%b 23.02 ± 4.967%c 
CD8+CD44highCD62Llow/neg 9.17 ± 1.38% 7.7 ± 0.82% 37.8 ± 7.1%b 12.03 ± 0.78%c 
T CellsWT BSA5-LO−/− BSAWT CFAgs5-LO−/− CFAgs
CD4+CD44highCD62Llow/neg 13.8 ± 2.24% 18.29 ± 1.68% 47.50 ± 6.19%b 23.02 ± 4.967%c 
CD8+CD44highCD62Llow/neg 9.17 ± 1.38% 7.7 ± 0.82% 37.8 ± 7.1%b 12.03 ± 0.78%c 
a

WT and 5-LO−/− mice were immunized with CFAgs and BSA following the challenge with a lethal dose of H. capsulatum. At day 7 postinfection with H. capsulatum, the lungs were analyzed, and the presence of memory T cells was determined. The results were calculated as percentage of CD4+ or CD8+ T cells with memory phenotype (CD4+CD44highCD62Llow/neg or CD8+CD44highCD62Llow/neg). Data represent mean ± SEM of six animals per group. Data from one of three experiments are shown.

b

p < 0.05 compared with WT BSA animals.

c

p < 0.05 compared with WT CFAg animals.

FIGURE 4.

Role of LTs in the recruitment of memory CD4+ T cells in the lung. Percentage of CD4+ T in the lungs expressing a memory phenotype (CD44high/CD62Llow/neg). Lungs of uninfected mice and CFAg- and BSA-immunized mice rechallenged with lethal dose of H. capsulatum were obtained, as described in Materials and Methods. The lymphocyte population was gated by forward/side scatters. Dot plots show a representative analysis from one of six mice. Results are from one experiment and are representative of three independent experiments.

FIGURE 4.

Role of LTs in the recruitment of memory CD4+ T cells in the lung. Percentage of CD4+ T in the lungs expressing a memory phenotype (CD44high/CD62Llow/neg). Lungs of uninfected mice and CFAg- and BSA-immunized mice rechallenged with lethal dose of H. capsulatum were obtained, as described in Materials and Methods. The lymphocyte population was gated by forward/side scatters. Dot plots show a representative analysis from one of six mice. Results are from one experiment and are representative of three independent experiments.

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To determine whether the pharmacologic inhibition of LT biosynthesis affected the generation/activation/migration of memory T cells to the infection site, C57BL/6 mice were treated with MK886 (FLAP inhibitor) only during the immunization or only during postinfection periods. Consistent with previous results (29), BSA-immunized mice succumbed by 14 days postinfection, whereas 72% of those immunized with CFAgs survived until day 60 postinfection.

Inhibition of LT biosynthesis by MK 886 during immunization (CFAgs + MK886Immunization) only partially affected the generation of the adaptive immune response. However, when the animals received MK886 postinfection (CFAgs + MK886Postinfection), the activation and/or migration of cells to the site of infection were abolished, resulting in 100% mortality (Fig. 5). Thus, these data suggest that in addition to impairment of the generation of memory T cells, the inhibition of LT synthesis abolished the migration and/or activation of memory CD4+ and CD8+ T cells.

FIGURE 5.

Effect of inhibition of LTs in the generation or activation of memory T cells. Survival (A) and fungal burden in lungs (B) and spleen (C) of CFAg- and BSA-immunized C57BL/6 mice treated with MK 886 only during immunization (CFAgs + MK886Immunization) or when the animals received MK 886 only after the infection (CFAgs + MK886Postinfection) and rechallenged with lethal dose of H. capsulatum. Mice were monitored for 60 days. D–F, Cytokine levels were quantified in the lung homogenates (n = 6–7). Tissue samples were harvested at day 7 of H. capsulatum infection. Results represent mean ± SEM of a representative (of two) experiment. UD, Undetected. ∗, p < 0.05 vs uninfected; #, p < 0.05 vs BSA immunized; ξ, p < 0.05 vs CFAg immunized.

FIGURE 5.

Effect of inhibition of LTs in the generation or activation of memory T cells. Survival (A) and fungal burden in lungs (B) and spleen (C) of CFAg- and BSA-immunized C57BL/6 mice treated with MK 886 only during immunization (CFAgs + MK886Immunization) or when the animals received MK 886 only after the infection (CFAgs + MK886Postinfection) and rechallenged with lethal dose of H. capsulatum. Mice were monitored for 60 days. D–F, Cytokine levels were quantified in the lung homogenates (n = 6–7). Tissue samples were harvested at day 7 of H. capsulatum infection. Results represent mean ± SEM of a representative (of two) experiment. UD, Undetected. ∗, p < 0.05 vs uninfected; #, p < 0.05 vs BSA immunized; ξ, p < 0.05 vs CFAg immunized.

Close modal

In another set of experiments, lungs and spleen were harvested from immunized mice treated or not with MK886 on days 7 and 14 after infection, and the fungal burden was determined. We did not observe significant differences in the lung and spleen CFU 7 days postinfection (data not shown). However, on day 14 after infection, we found a significant decrease in the lung (p < 0.001) and spleen (p < 0.05) CFUs of CFAg-immunized mice when compared with BSA-immunized mice (Fig. 5, B and C). Fungal burden in lungs and spleen of CFAg-immunized animals that received only MK 886 during immunization (CFAgs + MK886Immunization) was similar to CFAg-immunized mice. However, the treatment of CFAg-immunized mice with MK 886 only after challenge with H. capsulatum (CFAgs + MK886Postinfection) resulted in significant increase in the lung CFU (p< 0.01) and spleen (p < 0.001) when compared with CFA-immunized mice (Fig. 5, B and C).

To determine whether the difference in the susceptibility is associated with the production of the cytokines, TNF-α, IFN-γ, GM-CSF, IL-4, and IL-10, we evaluate its production in the lungs of these animals. The levels of TNF-α were increased in the lung of CFAg-immunized mice and treated with CFAg-immunized + MK886Immunization mice, but not in the lung of BSA-immunized and CFAg-immunized + MK886Postinfection mice (Fig. 5,D). IFN-γ production was increased only in the lung of CFAg-immunized mice; however, treatment with MK 886 impaired the synthesis of this cytokine (Fig. 5,E). Moreover, except in the lung from BSA-immunized mice, the synthesis of GM-CSF was increased in all CFA-immunized mice that received or did not receive treatment with MK 886 (Fig. 5 F).

The levels of IL-10 and IL-4 were similar in immunized mice with CFAgs or BSA (data not shown).

It is becoming clear that LTs are essential to the host defense of a variety of pathogens (34). However, it is still unknown whether LTs participate in the generation of a secondary immune response during microbial infections. The present study demonstrates that protection of WT mice immunized with CFAgs against H. capsulatum infection was associated with increased LTB4 recruitment of memory T cells, and increased production of NO and IFN-γ. Moreover, there was a decreased production of TGF-β and IL-10 in the lungs of these animals in a manner dependent on LT production.

CFAg immunization conferred protection in WT mice, but not 5-LO−/− mice (Fig. 1,A). In addition, WT CFAg mice presented more than 10-fold (1.1 log10) less H. capsulatum CFU in the lungs when compared with other groups (WT BSA, 5-LO−/− BSA, and 5-LO−/− CFAgs) (Fig. 1 B). Accordingly, the lungs of WT-BSA mice presented ∼3-fold increase in LTB4 production when compared with uninfected mice. These augmented levels were even higher (∼4-fold) in WT CFAg mice, but not 5-LO−/− CFAgs, which correlates with the mortality data. Unexpectedly, in accordance with Bafica et al. (35), LTB4 production was detected in the 5-LO−/− groups. We do not know why such levels can be detected in our condition; we speculate that it might be some nondescribed metabolic pathway or if they represent some intrinsic error on the detection kit. These results suggest that LTB4 production is up-regulated during H. capsulatum infection and its production is associated with the protective immunity attained by CFAg immunization.

The role of NO in host resistance during murine histoplasmosis is well known (36, 37). In fact, LT inhibition in primary histoplasmosis infection decreases NO production and increases mice susceptibility (17). Our results demonstrated that WT CFAg mice were the only ones that survived the lethal inoculum used, and this protection was associated with a 4-fold increase of NO in their lungs (Fig. 5). However, 5-LO−/− CFAg mice presented lower levels of NO, which correlates with a lack of protection in these animals. Serezani et al. (15) showed that LTB4 induce NO secretion and inducible NO synthase expression in murine macrophages and NO inhibition abolished LTB4 effects on macrophage leishmanicidal activity.

It is known that H. capsulatum immunogens such as heat shock protein 60 (Hsp60) are protective and trigger protective immune response associated with CD4+ T cells and production of IFN-γ, IL-12, and IL-10 (38). Thus, we sought to investigate whether the absence of 5-LO affects survival of H. capsulatum-infected mice through the modulation of cytokines involved in the process of T cell activation by measuring cytokines levels. TGF-β and IL-10 are cytokines involved in the host immunological homeostasis, and also display potent immunosuppressive activities in both phagocytes and lymphocytes (39). We observed that the increased susceptibility of 5-LO−/− BSA and 5-LO−/− CFAg mice was associated with an up-regulation of TGF-β and IL-10, but not protected WT CFAg mice. In our model, we suggested that CFA immunization increases LTB4 production, which might impair excessive TGF-β production and leads to progressive deterioration of the protective immune response. However, the effect of LTB4 on TGF-β production remains to be determined.

Memory T lymphocyte confers protection in peripheral tissues during a secondary immune response to pathogens. We have previously demonstrated that DTH in histoplasmosis is associated with the degree of the immune response (29). In this study, we demonstrated that WT mice immunized with CFAgs exhibited a strong DTH reaction, and 5-LO−/− mice developed a smaller reaction when compared with WT mice (data not shown). These results suggest that products of the 5-LO pathway could be important mediators involved in the generation of memory cells, and consequently to the development of a protective immune response to H. capsulatum. When we evaluated whether the increased susceptibility of 5-LO-deficient animals could be attributed to altered proportions of memory cells in the lung, our results demonstrated that WT CFAg mice are able to recruit at least 2-fold more memory CD4+ T cells in the lungs after H. capsulatum infection compared with 5-LO−/− mice (Fig. 4). In keeping with these results, Gomez et al. (40) demonstrated H. capsulatum-derived rHsp60 increases memory Vβ6 and Vβ8 CD4+ T cells in infected mice. In addition, the depletion of Vβ8.1/8.2+ CD4+ T cell subpopulation abolished protection induced by H. capsulatum-derived rHsp60. Although CD8+ T cells appear not to be required to confer protection during murine histoplasmosis, they contribute to the optimal clearance of H. capsulatum in the organs (2). In addition, a CD8+-dependent immunity to H. capsulatum may be achieved by vaccination of CD4+-depleted mice (41). We have observed a 4-fold increase in CD8+ T cells of H. capsulatum-infected WT CFAg mice, in comparison with 5-LO−/− CFAg mice (data not shown). Recently, it has been shown that LTB4 is a chemoctactic factor for effector T cells (27, 28), but not naive T cells or central memory CD8+ T cells. However, the specific role of LTB4 in the recruitment of CD8+ T cells during pathogen infection is unknown. Our results demonstrate that the immunization of WT CFAg mice induces the generation and recruitment of memory CD4+ and CD8+ T cells to the infection site. Moreover, it seems that products of 5-LO activity (e.g., LTs) may affect the establishment of a protective response conferred by the immunization as well as recruitment and activation of memory T cells during subsequent infection. Furthermore, the migration of effector cells from WT mice into the inflamed peritoneal cavity is 3 times higher than that observed in BLT1-deficient mice (27, 28). The pharmacological approach using MK886 (a FLAP inhibitor) did not affect the generation of the adaptive immune response (CFAgs + MKImmunization), but rather the activation and/or migration of cells to the infection site. This assumption is supported by the same survival rate and fungal burden presented by WT CFAg mice as well as MK886-treated mice during the immunization period (CFAgs + MKImmunization). However, when mice were treated with MK886 after challenge with H. capsulatum (CFAgs + MKPostinfection), all animals displayed higher fungal dissemination and dramatic reduction of protective immunity. Moreover, such as observed in 5-LO−/− CFAgs, the inhibition of LTs by MK 886 postinfection with H. capsulatum resulted in the inhibition of protective cytokines, such as IFN-γ. These data strongly suggest that LT deficiency (both genetic and pharmacologic) during infection is more associated with impairment in the migration and/or activation of memory CD4+ and CD8+ T cells, than the generation of them.

The lack of 5-LO products in special LTs is associated with the inhibition of recruitment and/or activation of effector memory T cells during H. capsulatum infection, resulting in mortality of immunized mice and increased CFU in the lung and spleen. LTB4 affects many cells involved in both the innate and adaptative immune response. LTB4 enhances phagocytosis, microbicidal activity, as well as generation/modulation of chemoattractants, such as chemokines and cytokines. In addition, LTB4 enhances dendritic and T cell migration and activation and mounting of the immune response. Because LTB4 acts on both arms of the immune response and because it is rapidly produced during infections or inflammatory conditions (seconds to minutes), it is an important target for adjuvant studies.

We thank Marc Peters-Golden for valuable discussions and for helpful comments, and C. Lewis for revising the manuscript. We are grateful to Merck Frosst Canada for furnishing the MK 886.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (02/07849-7; 02/12856-2), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), from Brazil and American Lung Association.

3

Abbreviations used in this paper: LT, leukotriene; 5-LO, 5-lipoxygenase; AA, arachidonic acid; BHI, brain-heart infusion; BLT1, LTB4 receptor 1; CFAg, cell-free Ag; DTH, delayed-type hypersensitivity; FLAP, 5-LO-activating protein; Hsp, heat shock protein; i.t., intratracheal; KC, keratinocyte-derived chemokine; neg, negative; p.o., perorally; WT, wild type.

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