The Relative Susceptibility of Mouse Strains to Pulmonary Cryptococcus neoformans Infection Is Associated with Pleiotropic Differences in the Immune Response

Yeast strain and preparation of inocula for infection. Cryptococcus neoformans ATCC 24067 (ATCC, Manassas, VA) was used in all experiments. This strain has been extensively used in studies of pulmonary pathogenesis and was used here to maintain continuity with the prior literature (30). For mouse infection, the cells were grown in Sabouraud medium until late logarithmic phase, washed and suspended in phosphate-buffered saline (PBS), and counted with a hemocytometer. Yeast cells were suspended in PBS at a cell density that would provide a fixed number of cells in the volume that was used to infect the mice (50 μl for intratracheal [i.t.] infections and 100 μl for intravenous [i.v.] infection). Inocula were confirmed by plating on Sabouraud dextrose agar and counting CFU (Difco, Detroit, MI).

Mouse strains, Ab administration, and routes of infection.Female BALB/c and CBA/J mice (6 to 8 weeks old; Jackson Laboratories, Bethesda, MD) were used in this study. We chose female mice because this sex was previously used in immunology studies in our laboratory. For the Ab protection experiments, the protective immunoglobulin G1 (IgG1) monoclonal Ab (MAb) 18B7 (7) was administrated intraperitoneally 24 h prior to infection. MAb 18B7 was purified by protein A affinity chromatography (Pierce, Rockford, IL) following the manufacturer's recommendations. Different amounts of Ab (1, 0.1 or 0.01 mg) were injected in a total volume of 0.5 ml. i.t. infections were performed as described previously (20). For i.v. infections, 100 μl of fungal cell suspension was injected in the caudal vein of the mouse tail.

Organ fungal burden.Organ fungal burden was inferred from the number of CFU per organ on plates. Infected mice were sacrificed, and the lung, brain, and spleen were removed and homogenized in PBS buffer. Various dilutions of the organ homogenate were plated in Sabouraud plates, and after 2 days of incubation at 30°C, the number of colonies was enumerated.

Serum GXM measurement.The amount of GXM in serum was determined by capture enzyme-linked immunosorbent assay (ELISA) as described previously (8). Briefly, serum samples were treated with proteinase K to destroy associated Ab and interfering proteins (1 mg/ml, overnight at 37°C; Roche, IN). Ninety-six-well plates were coated with goat anti-mouse IgM (1 μg/ml; Southern Biotechnologies, Birmingham, AL) followed by capture Ab 2D10 (2 μg/ml) (48). Samples were added, and GXM of known concentration was used as a standard. Bound GXM was detected using IgG1 MAb 18B7 (2 μg/ml) and secondary goat anti-mouse IgG1 Ab conjugated to alkaline phosphatase (1 μg/ml; Southern Biotechnologies, Birmingham, AL). A 1-mg/ml concentration of p-nitrophenyl phosphate dissolved in substrate buffer (1 mM MgCl₂·6H₂O, 50 mM Na₂CO₃) was used for color development, and the absorbance was measured at 405 nm using a microplate reader after approximately 20 min of incubation at room temperature.

Ab measurement.Serum IgM and IgG to GXM were determined by ELISA as described previously (8). Total IgE levels were determined by capture ELISA using specific Abs (Pharmingen, San Diego, CA) according to the manufacturer's instructions.

Ab binding to C. neoformans cells in vivo.Mice were infected with C. neoformans strain 24067 as described above and sacrificed after 7 days, and cells were obtained as described previously (76). Abs bound to the capsule were then detected by immunofluorescence using secondary Abs to mouse IgG or IgM conjugated to tetramethyl rhodamine isothiocyanate (Southern Biotechnologies Associates, Birmingham, AL).

Cytokines.Cytokine and chemokine levels were determined in whole organ lysates. Briefly, whole lungs were excised from C. neoformans-infected mice and sham-infected controls and homogenized in 2 ml of PBS containing proteases inhibitors (Boehringer Mannheim, Indianapolis, IN). The lysates were centrifuged, and the supernatants were kept at −80°C until they were analyzed. Cytokine and chemokine levels were determined using the Pharmingen (San Diego, CA) ELISA kit according to the manufacturer's instructions.

FACS.Analysis of inflammatory cell content in the lung was performed by fluorescence-activated cell sorting (FACS) as described previously (44). Briefly, mice were infected with 10⁶C. neoformans cells i.t. or i.v., and after 7 days of infection, they were sacrificed. The lungs (i.t. infection) or spleens (i.v. infection) were isolated and homogenized. Lysates were then treated with collagenase A (1 mg/ml; Boehringer Mannheim, Chicago, IL) in digestion buffer (RPMI medium, 10% fetal calf serum) and incubated for 1 h at 37°C with occasional shaking. Cells were centrifuged and suspended in 0.17 M NH₄Cl to lyse erythrocytes for 10 min at 4°C. A 10-fold excess of RPMI solution was then added to make the solution isotonic, and the cells were collected by centrifugation and suspended in staining buffer (PBS, 1% fetal bovine serum). The number of cells was determined by counting in a hemocytometer. For FACS analysis, lung leukocytes (10⁶) were stained for 30 min on ice with 100 μl of one of the following Abs diluted in staining buffer: 2 μg of R-phycoerythrin-labeled anti-CD45⁺ per ml, 5 μg of fluorescein isothiocyanate (FITC)-labeled anti-mouse CD4⁺ per ml, 2 μg of Cy-Chrome-labeled anti-mouse CD8⁺ per ml, 5 μg of FITC-labeled anti-mouse CD19⁺ per ml, or 5 μg of FITC-labeled anti-mouse MAC-3 per ml (all MAbs were from Pharmingen, San Diego, CA.). The samples were washed twice in staining buffer and fixed in 1% paraformaldehyde. Stained samples were stored in the dark at 4°C until analyzed on a FASCalibur flow cytometer (Becton Dickinson, Mountain View, CA) with CellQuest software (Becton Dickinson). Live cells were gated as judged from forward and side laser scatter and CD45 staining.

In vivo phagocytosis.10⁷C. neoformans ATCC 24067 cells were injected i.t. into both CBA/J and BALB/c mice. After 2 h, the mice were sacrificed, and alveolar lavage was then performed 10 times. The cell suspension was allowed to attach to 96-well plates for 2 h, fixed with methanol, and then stained with Giemsa stain as described previously (77). The phagocytic index (PI) was expressed as the number of internalized C. neoformans cells per 100 macrophages counted.

Bronchoalveolar lavage, in vitro phagocytosis, and subsequent imaging.Mice were sacrificed by asphyxiation with CO₂, and their tracheas were cannulated with angiocaths (BD Biosciences, San Jose, CA). Lungs were lavaged 10 times with sterile PBS with 1 mM EGTA (Sigma-Aldrich, St. Louis, MO). Lavage fluids were pooled, and cells were collected by centrifugation. Red blood cells were lysed by incubation in 0.17 M NH₄Cl at 4°C for 10 min. Cells were washed with PBS and suspended in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA) with 10% heat-inactivated fetal calf serum (Gemini Bioproducts, Woodland, CA), 10% NCTC-109 medium (Gibco, Carlsbad, CA), and 1% nonessential amino acids (Mediatech Cellgro, Washington, DC). Cells were then plated on polylysine coverslip-bottom MaTek plates (Ashland, MA) at a density of 1 × 10⁵ per well in feeding medium and allowed to adhere at 37°C with 10% CO₂ for 1 h prior to incubation with C. neoformans for an additional 1 h and subsequent microscopic imaging. The medium was then removed from MaTek plates and replaced with fresh medium containing C. neoformans cells (C. neoformans-to-macrophage ratio of 5:1) along with 50 μg/ml MAb 18B7. Macrophages were incubated with a solution that contained the yeast cells, 0.3 μg/ml lipopolysaccharide (Sigma, St. Louis, MO), and 50 units/ml of murine IFN-γ for 60 min. After this period of time, plates were washed twice with fresh medium to remove extracellular C. neoformans cells. Plates were then replenished with 2 ml feeding medium, and macrophages with ingested yeast cells were imaged under the microscope for 24 h. Images were collected at a magnification of ×40 every 3 min using an Axiovert 200 M inverted microscope and photographed with an AxiocamMR camera controlled by the Axio Vision 4.4 software (Carl Zeiss Micro Imaging, NY). Movies were compiled and analyzed using the ImageJ software (W. S. Rasband, National Institutes of Health, Bethesda, MD) (http://rsb.info.nih.gov/ij/ , 1997 to 2006).

Statistics.CFU, cytokine and FACS data were analyzed using the t test or the Kruskal-Wallis test after assaying normality by using the Shapiro-Wilk test with the Unistat software for Excel (Unistat, Ltd., London, United Kingdom). Statistical differences in survival rates were assessed using log rank analysis with SPSS software (SPSS, Chicago, IL). Statistical differences in intracellular replication inside alveolar macrophages were determined according to Fisher's exact test following the guidelines described at http://www.exactoid.com/fisher/index.php?a = 11&b = 24&c = 1&d = 26.

Differences in survival between strains at different infection doses.Previous studies (29) and our own laboratory experience have shown great differences in the susceptibility of mouse strains to C. neoformans infection. We revisited this question by a preliminary study that compared the outcome of C. neoformans pulmonary infection in BALB/c, A/J, C57BL/6J, and CBA/J strains and found that the BALB/c and CBA/J strains were the least and most susceptible, respectively (Fig. 1A). Therefore, we hypothesized that comparison of the immune responses of these two different mouse strains after C. neoformans infection could reveal factors involved in the resistance or susceptibility to cryptococcosis.

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FIG. 1.

Survival of BALB/c and CBA/J mice after C. neoformans infection. (A) CBA/J (circles) or BALB/c (inverted triangles) mice were infected i.t. with 10⁵ (open symbols) or 10⁶ (closed symbols) yeast cells, and survival was monitored daily. Each group contained between 8 and 10 mice (P < 0.05). (B) CBA/J (dotted line) and BALB/c (solid line) mice were infected i.v. with 10⁵ yeast cells, and survival was monitored as for panel A. Each group contained between 8 and 10 mice.

Measurement of organ fungal burden revealed that the enhanced susceptibility of CBA/J mice to pulmonary infection was associated with greater extrapulmonary dissemination, as evidenced by higher CFU in brain than measured for BALB/c mice (Fig. 2). We considered that the higher brain and spleen CFU in CBA/J mice could reflect greater dissemination from the primary pulmonary infection or a less effective systemic immune response. To investigate these possibilities, we examined the relative susceptibility of these strains to i.v. infection. We reasoned that if the difference in susceptibility between CBA/J and BALB/c mice was due to a difference in extrapulmonary dissemination, then the strains should have comparable susceptibility to i.v. challenge. Using an i.v. challenge of 10⁵, BALB/c mice were equally susceptible as CBA/J mice (Fig. 1B). Analysis of organ fungal burden after i.v. infection revealed, as expected, dissemination of the yeast through all the organs cultured in both mouse strains (Fig. 2B); in some organs, such as the lung, there was a trend in the BALB/c mice for higher fungal burden, but this did not achieve statistical significance. Given comparable BALB/c and CBA/J susceptibility to systemic infection, the higher extrapulmonary CFU observed in CBA/J mice after pulmonary infection probably reflects increased dissemination from the lungs.

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FIG. 2.

Organ fungal burden after C. neoformans infection. CBA/J (open bars) and BALB/c (closed bars) mice infected with 10⁶ yeast cells i.t. (A) or i.v. (B) were sacrificed 14 days after infection, and the organ fungal burden was counted as indicated in Materials and Methods. Mean values and standard deviations from five different mice are plotted. Significant differences determined using a nonparametric test (Kruskal-Wallis test) are highlighted with an asterisk, and the P value is shown.

GXM levels in serum.Serum GXM levels positively correlated with host fungal burden. Hence, we measured GXM levels in serum after C. neoformans infection in both strains. After i.t. infection, there was a trend to find higher GXM levels in the more susceptible CBA/J mice (P = 0.6), suggesting a more pronounced disease (Fig. 3). However, when the mice were infected i.v., there were significantly higher GXM levels in the BALB/c strain (P = 0.027), consistent with the relatively equivalent susceptibility change of this strain to i.v. challenge.

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FIG. 3.

Serum GXM levels. Sera from mice infected i.t. or i.v. after 7 days of infection were obtained, and GXM levels were measured by capture ELISA as described in Materials and Methods. Significant differences determined using a nonparametric test (Kruskal-Wallis test) are highlighted with an asterisk, and P values are shown. Mean values and standard deviations from five different mice are plotted.

Ab responses in CBA and BALB/c mice.Ab responses to GXM after i.t. infection were higher in BALB/c mice than in CBA/J mice (Fig. 4). Both IgM and IgG to GXM were present in the sera of infected BALB/c mice (Fig. 4). When the mice were infected i.v., the Ab response to GXM was much lower than in the i.t. model, such that for BALB/c mice, Ab titers were barely above the detection limit of the method used, and no Abs to GXM could be detected in the sera of infected CBA/J mice (results not shown). Endogenously produced mouse Abs to C. neoformans have been reported to have immunofluorescence characteristics of nonprotective Abs with a punctate pattern (76). Hence, we studied the pattern of binding of serum Abs in these mouse strains. We found a great heterogeneity in the size of the cryptococcal cells isolated from lungs. C. neoformans cells recovered from the lungs of the resistant mouse strain (BALB/c) were coated with Abs that gave a diffuse annular pattern with some fluorescence dots. However, in the susceptible strain, the larger cells were not labeled, and only the small cells gave a punctate pattern (Fig. 5).

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FIG. 4.

Measurement of Ab levels in response to GXM. The serum samples described in legend for Fig. 3 (i.t. infected mice) were tested for levels of IgG (A) and IgM (B) to GXM by ELISA as described in Materials and Methods. Each line and symbol refer to the data obtained from each mouse.

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FIG. 5.

Fluorescence of Abs bound to C. neoformans cells in vivo. C. neoformans cells were obtained from the lungs of infected mice as described previously (76), and Ab binding pattern was observed by fluorescence using secondary goat anti IgG plus IgM tetramethyl rhodamine isothiocyanate-labeled Abs. The right panels show the fluorescence patterns of the cells shown in the left panels. The scale bar in left upper panel (10 μm) applies to the all of the pictures.

Inflammatory response. C. neoformans infection is associated with increased IgE levels (3, 59, 60). Given that IgE levels reflect the balance between Th1 and Th2 responses, we measured serum IgE before and after infection in both mouse strains. In BALB/c mice, the IgE levels dropped significantly after i.t. infection (around a 30-fold decrease; P = 0.01), while in the susceptible strain, these levels did not respond to the infection (result not shown). When the mice were infected i.v., the response of serum IgE levels differed for the BALB/c strain, such that for this strain total IgE levels in the serum increased after infection (threefold increase; P = 0.02), indicating a change in the Th1-Th2 response regulation. As in the i.t. model, CBA/J mice did not regulate IgE levels upon i.v. infection.

After i.t. challenge, interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), and IFN-γ increased in response to C. neoformans infection in lung extracts, but no difference in their regulation during infection was found between the two strains (Fig. 6). However, lung IL-10 and IL-12 levels were differently regulated in the two mouse strains. In the susceptible strain (CBA/J), IL-10 was up-regulated (P = 0.014), and IL-12 levels did not change after infection. In the resistant strain (BALB/c), IL-10 levels decreased (P = 0.05) and IL-12 increased (P = 0.02) with infection. To analyze the cytokine response to infection in the i.v. model, we measured cytokine levels in the spleen, since it was the organ macroscopically most affected as evidenced by its greater inflammation. We found no difference between the strains, except in MCP-1, which was moderately increased in BALB/c mice after infection. The lack of IL-10 and IL-12 regulation after i.v. challenging is consistent with the similar susceptibility shown by the two strains in this model.

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FIG. 6.

Cytokine and chemokine levels in target organs after i.t or i.v. infection. BALB/c and CBA/J mice (n = 5) were infected i.t. or i.v. with 10⁶C. neoformans cells (closed bars) or injected with PBS as a sham control (open bars). The mice were sacrificed after 7 days, and cytokine and chemokine levels in the lung or spleen homogenates were measured by capture ELISA as described in Materials and Methods. Asterisks denote statistical differences (P < 0.05) determined using the nonparametric Kruskal-Wallis test. Mean values and standard deviations from five different mice are plotted.

Cellular recruitment into the lung was measured by FACS using cell type-specific Abs to enumerate both the absolute number of cells and the relative frequency of the various cell types. For this experiment, we infected the mice, isolated the inflammatory cells from the lung, and detected the proportions of macrophages, CD4⁺ cells, and CD8⁺ cells by FACS (Fig. 7). The inflammatory infiltrate in the CBA/J strain manifested a decrease in the proportion of T cells in the lung, both CD4⁺ and CD8⁺, and an increase in the proportion of macrophages compared to that in the BALB/c strain (Fig. 7). We performed a similar experiment with mice infected i.v. When the two mouse strains were infected i.v. with C. neoformans, there was no difference in cell recruitment in the spleen, a finding consistent with the lack of difference in mortality between the strains in this condition.

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FIG. 7.

Cell recruitment after C. neoformans infection. Mice were infected as described for Fig. 6, and cell suspensions were obtained from the lungs (i.t. infection) or the spleens (i.v. infection) of infected and sham-infected animals. The proportions of CD4⁺ cells, CD8⁺ cells, and macrophages among the leukocyte population, determined by using the CD45⁺ marker, were obtained by FACS. Asterisks denote significant differences (obtained using Student's t test), and P values are shown. Mean values and standard deviations from five different mice are plotted.

In vivo phagocytosis and intracellular replication.We measured phagocytosis in vivo by evaluating the proportion of alveolar macrophages that contain ingested yeast cells. The PIs were similar in the two mouse strains (PI_CBA/J, 21 ± 12; PI_BALB/c, 20 ± 4 [P > 0.05]), suggesting that susceptibility differences were not associated with phagocytosis efficiency.

Recent studies demonstrate that differences in susceptibility between rats and mice correlate with differences in C. neoformans intracellular replication inside macrophages. Consequently, we evaluated intracellular replication of C. neoformans in alveolar macrophages isolated from CBA/J and BALB/c mice. For this purpose, we followed the intracellular fate of the yeast cells by live microscopy imaging for 24 h. We observed that replication occurred more frequently in macrophages from CBA/J mice (Table 1) than in those from BALB/c mice, where intracellular replication was observed in only a small percentage of the macrophages. Interesting, the level of yeast extrusion by the macrophages remained similar between macrophages from BALB/c and CBA/J mice (data not shown).

Efficacy of passive Ab efficacy in i.t. and i.v. models.Passive MAb administration can modify the outcome of cryptococcal infection in mouse models of cryptococcosis. Hence, we studied the effect of MAb 18B7 administration in the two most susceptible infection models observed for CBA/J and BALB/c mice: i.t. infection for CBA/J mice and i.v. infection for BALB/c mice. Administration of small amounts of MAb 18B7 (10 μg) to CBA/J mice after i.t. infection tended to enhance the disease (although the difference was not statistically significant), while large amounts of MAb (1 mg) prolonged survival compared to that of the nontreated mice (P = 0.044) (Fig. 8A). To assess the impact of passive Ab administration on the course of i.v. infection, BALB/c mice were infected with 10⁴, 10⁵, and 10⁶C. neoformans cells in the presence and absence of MAb 18B7. MAb administration, irrespective of Ab concentration, did not have any protective effect in BALB/c mice infected with 10⁵ and 10⁶ yeast cells (Fig. 8C and D). BALB/c mice infected with 10⁴ yeast cells and treated with 100 μg of MAb survived longer (median survival of 282 days) than mice treated with PBS or with 1 mg or 10 μg of MAb 18B7 (median survival of 153, 51, and 153 days, respectively) (P < 0.05). In fact, administration of 1 mg of MAb accelerated mortality (although this was not significant [P = 0.06]), and 10 μg of MAb did not have any effect on survival (Fig. 8B).

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FIG. 8.

Passive Ab administration to CBA/J or BALB/c mice. Mice were treated with different amounts of MAb 18B7 and then infected with C. neoformans strain 24067. (A) i.t. infection. CBA/J mice (n = 8 to 10) were treated with 10, 100, and 1,000 μg of MAb 18B7 and then infected with 10⁶C. neoformans cells. (B to D) Survival of 6- to 8-week-old female BALB/c mice following i.v. infection with 10⁴ (B), 10⁵ (C), and 10⁶ (D) C. neoformans strain 24067 organisms, respectively, and treatment with different amounts of MAb 18B7 (n = 5 for each experimental group).