Introduction

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Cryptococcosis is a systemic infection caused by the encapsulated yeast, Cryptococcus neoformans (1). Strong circumstantial evidence suggests that the organism, which is widely distributed in nature, infects via the respiratory tract. In susceptible individuals, invasion of the central nervous system may lead to the development of a chronic meningoencephalitis, the most common presenting manifestation of a cryptococcal infection. Although cryptococcosis may occur in individuals who are immunocompetent, the risk of the infection is clearly higher in individuals who are immunosuppressed. For example, individuals with AIDS are at high risk, indicating that CD4 T cell-mediated immunity is a critical component of an effective host response. The organism does not produce a known toxin and its pathogenicity is thought to involve several complementary mechanisms. The polysaccharide capsule is an important virulence factor (reviewed in Refs. 2, 3). The capsule interferes with phagocytosis and if a large excess of cryptococcal polysaccaride is injected intravenously into mice, it can also induce T cell suppression. In murine infection models, acapsular mutants are significantly less virulent than encapsulated strains. The potential importance of the capsule in virulence has been demonstrated using systemic models in which the organism is either introduced intravenously (4) or, less commonly, is inoculated via the respiratory tract, a model which more closely mimics the natural route of infection (8). However, an absolute role for the capsule in lung virulence has not been unequivocally shown, because the pulmonary infection model employed an acapsular strain obtained by inducing mutations in an encapsulated C. neoformans strain (9), a strategy that is conducive to generating multiple hidden mutations.

The role of the capsule in pulmonary cryptococcosis is best studied using congenic strain pairs that differ only in capsule forming ability. The strategy would preferably be done using a serotype A C. neoformans, because worldwide the majority of cases of cryptococcosis are caused by this serotype. Congenic strains can be created in several ways. A single capsule gene can be deleted from a wild type as has been reported by Chang and Kwon-Chung in a serotype D strain (6, 7). Conversely, an acapsular strain that carries a genetic lesion in one of the CAP genes can be corrected by gene replacement or complementation. The acapsular strain 602, a spontaneous mutant, carries a defect in the CAP64 gene that is essential for capsule formation (7). Strain 602 is not only devoid of capsule as detected by India Ink staining, but is serologically untypeable, a characteristic of a cryptococcal yeast lacking a polysaccharide capsule. When strain 602 was complemented with the CAP64 gene, the complemented strain, TYCC38-602, acquired a serotype A polysaccharide capsule, demonstrable in vitro and in vivo, and the ability to produce a lethal infection in mice following intravenous inoculation (7). In a separate report, restoration of the capsule of strain 602 restored its complement activating ability and inhibited its ability to cause a primed macrophage cell line to produce tumor necrosis factor (TNF)- (10). This latter observation is particularly important, because TNF- is a critical factor in the initiation of an effective cell-mediated immune response against C. neoformans in a murine pulmonary infection model (11) and in the maximal induction of the inducible nitric oxide synthase (iNOS) gene (12). NO, the product of iNOS induction in macrophages, is required to mediate effective lung clearance of C. neoformans in a murine pulmonary infection model (13).

We sought to determine whether acquisition of a capsule by 602 would increase virulence in a lung infection model and whether this might lead to a failure to develop cell-mediated immunity in the lung. We found that complementation of 602 with the CAP64 gene increased pulmonary virulence; i.e., the encapsulated transformant, TYCC38-602, grew more readily in the lungs of mice and was more likely than the parent 602 to disseminate to the brain. However, acquisition of a capsule did not suppress the development of early pulmonary cell-mediated immunity. Thus, both the parent 602 and the TYCC38-602 grew more readily in the lungs of T cell- depleted mice, indicating that both 602 and TYCC38-602 infection induced effective immune responses in infected immunocompetent mice. Interestingly, a plasmid control transformant (CIP3-602) grew less readily in the lungs than the wild type unencapsulated strain (602) and was cleared no more readily in immunocompetent compared with T cell- depleted mice. We speculate that the random nature of the insertion of the plasmid vector might have disrupted a virulence gene expressed in the parent strain 602 and thereby decreased the overall virulence of CIP3-602.

	Materials and Methods

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C. neoformans Strains

602 (acapsular mutant), CIP3-602 (plasmid control transformant), and TYCC38-602 (encapsulated transformant) have been previously described (7, 10). The encapsulated transformant TYCC38- 602 was produced by electroporating 602FO1 (a spontaneous ura5 mutant of 602) with a plasmid containing both the URA5 and the CAP64 gene. CIP3-602 was made by transforming 602FO1 with a plasmid vector containing only URA5. All three strains were maintained on yeast extract peptone dextrose (YEPD) slants and grown in YEPD broth (1% yeast extract, 2% peptone, 2% dextrose) for the inoculation protocol. The three strains grow equally well at 37°C in vitro and produce dark brown colonies on birdseed agar due to their production of melanin (data not shown).

Mice

C.B-17 mice were raised in the University of New Mexico Animal Resource Facility. Mice, 6 to 12 wk old, were housed in filter top cages in a pathogen-free environmental unit. Sterile food and water were given ad libitum. Sentinel mice from breeding colonies and experimental areas are routinely analyzed serologically and histologically for evidence of secondary infections. The UNM ARF is accredited by the American Association for Accreditation of Laboratory Animal Care, and all animal protocols were reviewed and approved by the UNM Institutional Animal Care and Use Committee.

Inoculation of Mice with C. neoformans Strains

Mice were inoculated intratracheally with 5 × 10⁵ organisms which were grown at 30°C in YEPD broth overnight and then in fresh broth for 5 h before dilution in saline and inoculation. Anesthetized mice were inoculated intratracheally by making a small incision to expose the trachea and inserting a curved needle through which 50 µl of the yeast suspension was delivered containing the desired amount of organisms. The incision was sealed with super glue.

Isolation of Lung and Lung-Associated Lymph Node Cells

Lung and lung-associated lymph node (LALN) cells were isolated from each infected mouse as previously described (14). Mice were pretreated with heparin intraperitoneally (150 U; ELKINS-SINN, Inc., Cherry Hill, NJ) 10 min before being killed. The pulmonary vasculature was perfused with sterile saline to eliminate peripheral blood cells, and the lungs were removed, minced, and incubated with collagenase (0.7 mg/ml in RPMI with 5% fetal bovine serum (FBS); Boehringer Mannheim Biochemicals, Indianapolis, IN) and DNase (30 µg/ml Type IV bovine pancreatic DNase I; Sigma Chem. Co., St. Louis, MO) for 90 min at 37°C. Digested lungs were tapped through a wire mesh. Large particulate matter was removed by passing the cell suspension through a loose nylon wool plug. Cells were washed twice with Hanks' balanced salt solution (HBSS) and resuspended in RPMI with 5% FBS. Red cells were lysed, if necessary, using an ice-cold isotonic 0.14 M ammonium chloride solution (pH 7.4). Live cells were counted on a hemocytometer and assessed as those cells that exclude Trypan blue. Cells (2.5 × 10⁴) were deposited on glass slides using a cytocentrifuge and stained with Diff-Quik (VWR Scientific Products, San Francisco, CA). The numbers of macrophages, lymphocytes, neutrophils, and eosinophils were determined from each mouse using standard morphologic criteria. For cytokine secretion studies, lung cells were spun through a 30% Percoll/phosphate-buffered saline solution (Pharmacia, Piscataway, NJ) to eliminate red cell ghosts and cellular debris from the lung cell preparation. Cells were resuspended in 5% FBS/cRPMI and incubated on 100 mm² tissue culture plates for 2 h at 37°C, 5% CO₂ to remove adherent cells. Nonadherent cells were collected and resuspended at 5 × 10⁶/ml in culture medium (RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM non-essential amino acids [all from Gibco BRL Life Technologies, Grand Island, NY], 5 × 10⁵ M 2-mercaptoethanol [Eastman Kodak, Rochester, NY] and 2 µg/ml amphotericin B [Sigma; to block cryptococcal growth in culture]). All lung cell cultures were supplemented with 1 µg/ml indomethacin (Sigma), and 250 U/ml catalase (Worthington Biochem, Lakewood, NJ) to avoid the effects of prostaglandins and oxygen radicals on suppression of lymphocytes in cultures. To prepare LALN cells for cytokine analysis, LALNs were removed from each mouse and disrupted between two sterile, frosted glass slides to obtain single cell suspensions. The LALN cells were washed with HBSS, enumerated, and resuspended in culture media at 5 × 10⁶/ml. Lung and LALN cells were cultured in duplicate in medium alone or stimulated with 5 µg/ml concanavalin A (Con A; Sigma). Anti-interleukin (IL)-4 receptor (1 µg/ml; Genzyme, Cambridge, MA) was included in all cultures to prevent secreted IL-4 from being bound by IL-4 receptor-positive T cells in the cultures. Supernatants were collected after 48 h of culture and analyzed for cytokine content.

Determination of C. neoformans Colony-Forming Units per Organ

Lungs, brains, spleens, and livers were removed from each mouse and homogenized in sterile water. Serial dilutions of these homogenates were plated in a volume of 50 µl on Sabarouds-Dextrose agar (Becton-Dickinson, San Jose, CA). Plates were incubated at 30°C for 48 h, and the dilution yielding between 10 and 75 colonies was enumerated and converted to colony-forming units (cfu)/organ. In some instances, lung cfu per mouse was determined by plating serial dilutions of collagenase-digested lung cells on Sabarouds-Dextrose agar in a similar manner. The assay is designed so that limit of detection of lung cfu was 100-1,000 cfu and that of brain cfu was 100 cfu. Therefore, when homogenates of either organ failed to produce any colonies, the limit of detection (log 3 or log 2) was entered for statistical comparisons.

Depletion of T Lymphocytes

Some C.B-17 mice were depleted of CD4+ and CD8+ T cells before inoculation with C. neoformans. Mice were injected intraperitoneally with 0.5 mg anti-CD4 (GK1.5 ascites) and 0.5 mg anti-CD8 (YTS169.4 ascites) on Day -1 relative to intratracheal inoculation. Control mice received either no injection, 1 mg rat IgG2b (SFR8.B6 ascites), or saline intraperitoneally. On Days 7, 14, and 28 after intratracheal inoculation with C. neoformans, mice received intraperitoneal injections of 0.25 mg anti-CD4 plus 0.25 mg anti-CD8 or 0.5 mg control IgG2b. All ascites were prepared from injection of SCID mice with the appropriate clones, a service provided by TSD Bioservices, Germantown, NY. Efficacy of T cell depletion was analyzed by staining collagenase-digested lung cells with affinity purified anti-CD4-fluorescein isothiocyanate (RM4.4, rat IgG2a), anti-CD8-PE (53-6.7, rat IgG2a), and anti- CD3-cychrome (145-2C11, hamster Ig). Monoclonal antibodies were purchased from BD PharMingen (San Diego, CA). Three-color immunofluorescence was evaluated on a Becton-Dickinson FACSCalibur and data were analyzed using Cell Quest software.

Cytokine Enzyme-Linked Immunosorbent Assays

Cytokines were analyzed using a two-site sandwich enzyme-linked immunosorbent assay (ELISA) as previously described (14). Capture mAbs for IL-4 (11B11), IL-5 (TRFK5), and interferon (IFN)- (R46A2) were obtained from BD PharMingen and bound to ELISA plates diluted in 0.1 M Na₂HPO₄ solution (pH 9.0). Nonspecific binding was blocked with a 1% bovine serum albumin (BSA)/PBS solution. Biotinylated detection mAbs (BD PharMingen) included: anti-IL-4 (BVD6-24G2), anti-IL-5 (TRFK4), and anti-IFN- (XMG1.2). Streptavidin-horseradish peroxidase (1 mg/ml) in blocking buffer was added to detect bound cytokines in the assays and developed using an ABTS (azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) solution and the O.D. at 405 nm was determined. Cytokines were quantified by comparison to standard curves using recombinant IL-4, IL-5, and IFN- (BD PharMingen). An internal standard was included to monitor reproducibility of ELISAs using recombinant IL-4, IL-5, and IFN- obtained from Genzyme. Detection limits for each cytokine were assigned as the lowest concentration in the linear portion of the standard curve, generally between 31 and 1,000 pg/ml.

Statistical Analysis

Differences in all measured variables between mice were analyzed using analysis of variance (ANOVA) statistics using the Fisher's post-hoc test when three groups were being compared or by unpaired two-tailed t tests when two groups were being compared (StatView software; SAS Institute, San Francisco, CA). Values of P  0.05 were considered significant for all comparisons.

	Results

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An Encapsulated Transformant, TYCC38-602, Is More Virulent than an Acapsular Parent Strain Following Intratracheal Deposition

We have previously used an intratracheal inoculation to compare resistance against developing a cryptococcal lung infection in various inbred mouse strains. We selected the C.B-17 mouse strain for the current study to determine if gain of capsule by 602 increased its virulence in an intratracheal inoculation model, because in previous studies this strain developed the greatest acquired resistance to pulmonary crytococcal infection (14). We inoculated 5 × 10⁵ cfu of the encapsulated transformant TYCC38-602, the parent 602, and a plasmid control transformant, CIP3-602, via the trachea. All pulmonary infections were well controlled by Day 42 (Figure 1A). However, both unencapsulated strains were cleared more readily than the encapsulated transformant TYCC38-602 before this time. After an initial decrease in the numbers of yeast deposited, TYCC38-602 replicated in the lungs between Days 14 and 28. After 28 d, cfu gradually dropped to levels similar to those of the 602 and CIP3-602. Interestingly, at Days 7 and 14, 602-infected mice had more lung cfu than CIP3- 602-infected mice. However, the average cfu at deposition was also lower, raising the issue of whether the lowered deposition accounted for the lower cfu at Days 7 and 14 of CIP3-602. After Day 14, lung cfu of both acapsular organisms were the same.

View larger version (12K): [in this window] [in a new window]   Figure 1.   Pulmonary (A) and brain (B) cfu in C.B-17 mice infected via the intratracheal route with the encapsulated transformant TYCC38-602 (closed circles), the acapsular parent strain 602 (X), or the plasmid control transformant CIP3-602 (open circles). C.B-17 mice were infected with 5 × 105 organisms and organs were harvested and analyzed for cfu as described in MATERIALS AND METHODS. All three groups of infected mice differed from one another in pulmonary cfu (two-way ANOVA). TYCC38-602-infected mice differed from both acapsular yeast infected mice in brain cfu, whereas the latter two groups of infected mice were not different from each other (one-way ANOVA). Individual times at which TYCC38-602 cfu were higher than both of the other two strains is indicated by an asterisk (P < 0.05, two-tailed t test). Times when 602 cfu were higher than CIP3-602 cfu are indicated by a t. The limit of detection of lung cfu was 100-1,000 cfu and that of brain cfu was 100. Therefore, when homogenates of either organ failed to produce any colonies, the limit of detection (log 3 or log 2) was entered for statistical comparisons. These data represent three different experiments and 6-16 mice/time point.

Similarly, extrapulmonary cryptococcal growth, as detected by cfu in the brains of infected mice, occurred in 40 of 68 TYCC38-602-infected mice studied throughout 42 d of infection (Figure 1B). There was no significant difference in brain cfu in TYCC38-602-infected mice between the five time points measured. Between 50 and 80% of mice exhibited brain dissemination at each time point. In contrast, only 3/68 mice infected with 602 and 1/67 mice infected with CIP3-602 demonstrated brain cfu at any of these time points. We speculated that following lung deposition, dissemination to the brain generally failed to occur in the mice infected with the acapsular yeast. However, we cannot exclude the possibility that acapsular organisms that escaped from the lung failed to grow in the brain.

TYCC38-602 Evokes a Greater Cellular Response in LALNs and Lung than 602 and CIP3-602

The growth curve of TYCC38-602 suggested that acquired immunity might have developed by Day 28 and, thus, might be expressed as an increased inflammatory infiltrate at that time. We previously demonstrated that preceding the development of pulmonary immunity to a strain D serotype (specifically 52D, which is American Type Culture Collection #24067; ATCC, Manassas, VA), LALNs increased in size and demonstrated the development of a Th1-mediated immune response as measured by LALN cellular IFN- secretion and the absence of IL-4 and IL-5 secretion (15). Furthermore, by Day 14, lung cell numbers were also increased by the infiltration of inflammatory cells, including macrophages and lymphocytes, and cultures of lung cells secreted IFN-. Therefore, LALN and lung cells were collected from mice infected with the three cryptococcal strains, and total cells were enumerated (Figures 2A and B), differential counts performed, and cytokine secretion measured. Both lung and LALN cellularity differed between all the strains as measured by ANOVA. In general, organ cellularity was highest in mice infected with TYCC38-602, particularly over the first 5 wk of infection. The asterisks indicate time points at which TYCC38-602-induced cellularity was significantly greater from that induced by either of the acapsular strains, whereas the percent sign indicates time points at which it was greater than CIP3-602-induced cellularity only. At Day 42 of infection, 602-infected mice had significantly more LALN and lung cells than CIP3-602-infected mice, but those levels were equivalent to those observed in TYCC38-602-infected mice (shown by #).

View larger version (24K): [in this window] [in a new window]   Figure 2.   Lung (A) and lung-associated lymph node (LALN; B) cell numbers after intratracheal inoculation with acapsular parent strain 602 (open bars), plasmid control transformant CIP3-602 (gray bars), and encapsulated transformant TYCC38-602 (black bars). All three groups of infected mice differed from one another in LALN and pulmonary cellularity (two-way ANOVA). Time points at which significantly higher lung and LALN cellularity was observed following intratracheal inoculation with TYCC38-602 as compared with inoculation with either of the two acapsular strains are indicated by an asterisk (P < 0.05, by a two-tailed t test). Time points at which organ cellularity in TYCC38-602-infected mice were significantly greater than in mice infected with CIP3-602, but not 602, are indicated by a % sign. Time points at which organ cellularity was increased in 602-infected mice as compared with CIP3-infected mice are indicated by a # sign. These data represent two separate experiments and 9-10 mice/time point.

The types of cells that infiltrated the lungs were evaluated (Figures 3A-3D). As shown, macrophages and lymphocytes were the major infiltrating cell, particularly in the TYCC38-602-infected mice, but small numbers of polymorphonuclear leukocytes (PMNs) and eosinophils were also present. At Day 28, when clearance in the TYCC38- 602-infected mice began, increased inflammatory cells of all types were present in the lungs of the encapsulated yeast-infected mice as compared with mice infected with the acapsular strains. 602-infected mice recruited statistically more lymphocytes and PMNs to the lung as compared with CIP3-602-infected mice, but equivalent numbers of macrophages and eosinophils. The increase in PMNs recruited by 602-infected as compared with CIP3-602- infected mice was statistically significant by ANOVA (P = 0.0384) only when the entire time course of infection is considered rather than any individual time points (P > 0.05 at all days of infection when comparing PMN numbers in 602- and CIP3-602 infected mice).

View larger version (32K): [in this window] [in a new window]   Figure 3.   Composition of lung cells after intratracheal inoculation with acapsular parent strain 602 (open bars), plasmid control transformant CIP3-602 (gray bars), and encapsulated transformant TYCC38-602 (black bars). A significant increase in macrophages, lymphocytes, and eosinophils in the lung occurred following intratracheal inoculation with TYCC38-602 when compared with the two unencapsulated strains (two-way ANOVA). Both TYCC38-602- and 602-infected mice recruited significantly more neutrophils to the lungs as compared with CIP3-602-infected mice. Significantly more lymphocytes were recruited to the lungs of 602-infected mice as compared with CIP3-602-infected mice. Individual times when cellular increases due to TYCC38-602 inoculation were significantly greater than in mice infected with either of the other two strains are indicated by an asterisk (P < 0.05, t test). Time points at which inflammatory cell recruitment in TYCC38-602-infected mice differed only from CIP3-602-infected mice are indicated by a % sign. Time points at which 602-infected lung cell recruitment differed from that of CIP3-602 infected mice are indicated by a # sign. These data represent one experiment and 2-5 mice/time point, and are representative of two additional experiments.

LALN and lung cells were evaluated for their capability to secrete the Th1 cytokine, IFN-, and the Th2 cytokines, IL-4 and IL-5. Overall, during the course of the infection, lung cells from both TYCC38-602- and 602-infected mice secreted statistically increased levels of IFN- and IL-4, but not IL-5, as compared with lung cells from CIP3-602-infected mice as analyzed by two-way ANOVA comparing strain of organism and day of infection (Figures 4A-4C). The days when the values of cytokine secreted by lung cells from CIP3-602-infected mice were significantly different from lung cells harvested from mice infected with either of the other two strains are indicated by symbols in Figure 4. Nevertheless, the levels of IFN- secreted by lung cells were less than that reported previously in mice infected with strain 52D, a strain much more readily cleared by C.B-17 mice than the TYCC38-602 strain (15). LALN cells secreted very low levels of all three cytokines regardless of the strain of yeast with which they were infected, and no differences were detected among strains (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 4.   Production of IL-4, IL-5, and IFN- by concanavalin A-stimulated nonadherent lung cells after intratracheal inoculation with the acapsular parent strain 602 (open bars), the plasmid control transformant CIP3- 602 (gray bars), and the encapsulated transformant TYCC38-602 (black bars). Lung cells harvested from CIP3-602-infected mice secreted significantly lower levels of IL-4 and IFN- than mice infected with either of the other two cryptococcal strains when evaluating the whole 42-d course of infection (two-way ANOVA). Individual times when the CIP3-602-infected mice secreted levels of IL-4 and IFN- which were significantly less than both of the other two strains are indicated by an asterisk. Times when cells from CIP3-602-infected mice secreted less cytokine than cells harvested only from TYCC38-602-infected mice are indicated by a % sign. Times when secretion from cells from CIP3-602-infected mice was less than from cells harvested only from 602-infected mice are indicated by a # sign. Spontaneous secretion of IL-4 and IFN- from lung cells cultured in media alone was not significantly different when comparing the three groups of infected mice by two-way ANOVA (data not shown). Levels of IL-4 and IFN- secreted were never above 1.2 and 1.5 ng/ml, respectively, in the absence of Con A stimulation. In contrast to secretion of IL-4 and IFN-, Con A-stimulated lung cells from mice infected with all three strains of cryptococci secreted similar amounts of IL-5. On Days 7 and 14, however, spontaneous secretion of IL-5 by lung cells was significantly higher in TYCC38-602-infected mice when compared with the other two groups (data not shown). Levels of spontaneously secreted IL-5, however, never exceeded 3.5 ng/ml. These data represent two experiments and 3-10 mice/time point.

T Cell Depletion Enhances Cryptococcal Lung Growth in Both TYCC38-602- and 602-Infected Mice, but Increases Brain Dissemination Only in TYCC38-602-Infected Mice

In murine pulmonary infection models, both CD4 and CD8 T cells are required for the clearance of low-virulence C. neoformans from the lungs of resistant mice. We determined whether the clearance that began at Day 28 in TYCC38-602-infected mice required T cells and whether the maintenance of low to absent cfu in the lungs and brains of 602- and CIP3-602-infected mice might also require CD4 and CD8 T cells. C.B-17 mice were treated with antibodies against both CD4 and CD8 one day before intratracheal inoculation of 602, CIP3-602, or TYCC38-602 to deplete T cells. Antibody treatments were repeated at Days 7, 14, and 28. Flow cytometric analysis of lung T lymphocytes at 7, 28, and 42 d after infection showed that depletion was nearly complete, although the total number of CD3+ T cells was returning to baseline levels by Day 42 (about half of control treated mice in 602- and CIP3-602-infected mice and about one-third of control in the TYCC38-602-infected mice [data not shown]). Not surprisingly, as noted for total lymphocyte recruitment (Figure 3B), TYCC38-602-infected mice that received control antibody recruited more T cells to the lung than did either of the other two groups of control antibody-treated infected mice (data not shown).

CD4/CD8 T cell-depleted C.B-17 mice infected with both the encapsulated transformant and the parent 602 failed to control the growth of the organism in the lungs (Figures 5A and 5C). In contrast, in CIP3-602-infected mice, T cell depletion had no effect on lung growth (Figure 5B). Notably, even though both 602- and CIP3-602-infected mice received similar depositions of the organisms, there were fewer CIP3-602 organisms on Day 7 in the lungs (similar to data shown in Figure 1). In CD4 and CD8 T cell- depleted mice, brain cfu were the same in the 602 and CIP3-602-infected mice as compared with control inoculated mice, but there was a small, but significant, increase in brain cfu in the TYCC38-602-infected mice. In applying the post hoc test, however, the difference was only significant at Day 28. This observation may be related to the higher lung cfu at Day 28 in the TYCC38-602-infected mice and more dissemination from the lungs. Neither control antibody nor diluent injections depleted T cells (data not shown) nor affected the lung clearance or brain dissemination of TYCC38-602 (Figures 5C and 5F).

View larger version (23K): [in this window] [in a new window]   Figure 5.   Effects of T cell depletion on pulmonary (A-C) and brain (D-F) cfu after intratracheal infection with acapsular parent strain 602 (A and D), plasmid control transformant CIP3-602 (B and E), or encapsulated transformant TYCC38-602 (C and F). Mice received intraperitoneal injections of anti-CD4 and anti-CD8 antibodies (open circles), isotype control antibody (closed circles), saline only (open squares, applicable only to the TYCC38-602 studies), or were left uninjected (closed squares, applicable only to the TYCC38-602 studies) as described in MATERIALS AND METHODS. The data represent two experiments in the case of 602 and CIP3-602 and one experiment using TYCC38- 602. The limit of detection of lung cfu was 1,000 cfu and that of brain cfu was 100. Therefore, when homogenates of either organ failed to produce any colonies, the limit of detection (log 3 or log 2) was entered for statistical comparisons. T cell depletion resulted in decreased lung clearance of 602 and TYCC38-602 and increased brain dissemination of TYCC38-602 as revealed by two-way ANOVA (P < 0.05). The asterisk indicates the time points in which the clearance was statistically different between the treated and control groups in each of the six graphs (n = 5-14/time point, except Day 0, when n = 3).

It was likely that the cause for the decreased lung resistance in CD4/CD8 T cell-depleted C.B-17 mice infected with TYCC38-602 and 602 related to a decreased ability of the immune suppressed mice to recruit effector cells to their lungs. Accordingly, lungs were analyzed for numbers of total infiltrating macrophages, lymphocytes, neutrophils, and eosinophils at 7, 28, and 42 d after infection (Figures 6A-6C). As expected, total lymphocytes were decreased at Days 7, 28, and 42 in CIP3-602- and TYCC38-602-infected mice with a trend to reduction at Days 7 and 28 in the 602-infected mice. In T cell-depleted, TYCC38-602-infected mice, there was a trend toward fewer macrophages and neutrophils recruited to the lungs early in infection; and this difference was significant for macrophages at Days 28 and 42 and for PMNs at Day 42 (Figure 6C). A small, but significant, decrease in eosinophils also occurred at all three times in T cell-depleted TYCC38-602-infected mice.

View larger version (18K): [in this window] [in a new window]   Figure 6.   Composition of lung inflammatory cells in control antibody treated (open bars) and anti-CD4 and anti-CD8 treated (filled bars) C.B-17 mice after intratracheal inoculation with acapsular parent strain 602 (A), plasmid control transformant CIP3-602 (B), or encapsulated transformant TYCC38-602 (C). Mice were treated with isotype control antibody (open bars) or anti-CD4 and anti-CD8 antibodies (closed bars). Increases in macrophages, lymphocytes, neutrophils, and eosinophils occurred in the lungs of control mice as compared with T cell-depleted mice, particularly in the TYCC38- 602 infected mice. Significant differences in lung cell numbers between T cell-depleted and control antibody treated mice are indicated by an asterisk (P < 0.05). These data represent two experiments and 7-11 mice/time point.

	Discussion

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The most important observation of these studies is that encapsulation gives C. neoformans a growth advantage in the murine lung. A second conclusion is that encapsulation facilitated brain colonization by cryptococci, which supports previous work (6, 7). However, the current study extends the earlier studies which used an intravenous inoculation, because the route of inoculation was via the lung, which more closely mimics the natural route of infection. Indeed, it is possible that the mechanisms in the earlier studies for increased virulence and death of the mice infected with the encapsulated organisms were different from those described here. That is, the increased growth in the brain of the TYCC38-602-infected mice in the current studies could have reflected the increased dissemination from lungs more heavily burdened with the yeast rather than any effect of capsule on host resistance in the brain. A third conclusion is that for both the encapsulated transformant and the unencapsulated parent strain yeast, mice required T cells to optimally control growth of the organism within the lungs. However, despite identical yeast burdens at Days 7 and 42 in TYCC38-602- and 602-infected mice, it took longer for 602 to reach the higher lung burden at Day 42 than for TYCC38-602 (see Figure 5, Day 28). This finding suggests that even in the absence of T cells, the lack of a capsule results in a less virulent organism, which corresponds with clinical observations that even in profoundly immunosuppressed people with AIDS, unencapsulated cryptococcal infections are unusual (16).

As discussed above, it is unclear whether the increased brain burden in the lung infection with the encapsulated transformant related to increased dissemination from the lung during the course of the infection or to reduced resistance within the brain following an initial spread after the intratracheal infection or both. Extrapulmonary dissemination is a function of an organism's ability to cross the bronchoalveolar epithelium, enter lymphatics for subsequent entry into the circulation, or directly invade the blood stream and then escape intravascular mechanisms that kill the yeast. In regard to intravascular mechanisms for killing cryptococci, the capsule activates complement and facilitates leukocyte uptake and oxidative cytotoxicity (17). Both encapsulated and acapsular organisms are readily killed by neutrophils in the presence of serum that contains active complement (21). However, C5 is required for killing to occur with encapsulated, but not acapsular strains, indicating that innate mechanisms are more stringent for encapsulated yeast killing by a complement-mediated neutrophil mechanism (21). It seems most likely that the increased brain burden in TYCC38-602-infected mice, as compared with those mice infected with acapsular strains, reflects multiple mechanisms, i.e., increased yeast in the immunocompetent lung with continual escape, a less efficient complement-mediated killing mechanism within the vascular compartment, and some poorly understood failure of cell-mediated immunity in the brain.

An important question is: What is the role of capsule in interfering with expeditious clearance of the organism in the lung? The polysaccharide capsule has the ability to prevent leukocyte emigration (22) and phagocytosis (23). A potential mechanism for decreasing emigration of leukocytes relates to the capacity of the polysaccharide to cause shedding of L-selectin and the TNF- receptor on leukocytes and/or endothelial cells (24). Notably in the current studies, the encapsulated transformant recruited increased leukocytes as compared with the unencapsulated strains, but this might have related to more local lung damage with enhanced survival of the organism counteracting the suppression of recruitment. Phagocytosis may be inhibited in vivo, because capsular polysaccharide (i) depletes complement (25), (ii) decreases antibody formation (26), and (iii) generates a high negative charge on the yeast surface (29). The decreased phagocytosis can inhibit antigen uptake by antigen presenting cells (30) and thus decrease the development of cell-mediated immunity. Finally, once sufficient polysaccharide from an ongoing infection is generated in vivo, it can initiate a T cell suppressor network with the capacity to prevent the development of cell-mediated immunity and to suppress the effector limb of the immune response (31).

A related question is: What is the role of T cells in mediating pulmonary clearance of C. neoformans? Lung clearance of strain 52D required CD4 and CD8 T cells (35) and depended upon an effective Th1 T cell response and IFN- (36). Macrophage recruitment can be mediated by T cells, and it has been shown that inhibiting macrophage recruitment interferes with cryptococcal clearance from the lungs (37). Furthermore, pulmonary clearance in C.B-17 mice infected with encapsulated strain 52D corresponded to the T cell-mediated recruitment and activation of macrophages to make NO (13). Nonetheless, the specific mechanisms by which lung clearance is mediated remains uncertain.

The current studies raise the issue of what immune-mediated mechanism resulted in inhibiting intrapulmonary growth of the acapsular 602 strain, particularly in view of the relative paucity of inflammation in the immunocompetent, 602-infected mice. The parent strain 602 was a clinical isolate from a case of cryptococcal meningoencephalitis (38). Isolation of acapsular mutants from clinical cases is quite rare, suggesting that 602 retains one or more important virulence factors not necessarily present in most other acapsular C. neoformans strains created in the laboratory. In our pulmonary infection model with strain 602, fewer cells were recruited relative to the numbers recruited by the encapsulated transformant, but depletion of CD4 and CD8 T cells still adversely affected lung clearance. This observation suggests that T cells recruited to 602-infected murine lungs either had a direct effect on cryptococcal growth or activated resident macrophages to clear the infection by secreting IFN-. It has been well documented that T lymphocytes have the capacity to directly mediate stasis of cryptococci (39, 40). A role of resident macrophages playing a role in clearance could be tested by showing that resident macrophages from 602-infected mouse lungs express iNOS and inhibit the organism in vitro. Furthermore, blocking NO production would be expected to inhibit clearance of the 602 strain.

A surprising finding was that early lung clearance (Days 7 and 14) of the parent 602 strain was slower than clearance of the plasmid control strain, CIP3-602, in the experiments done in immunocompetent mice (see Figures 1 and 5). This difference could have been due to an unintended average smaller inoculum size of CIP3-602 at Day 0 in Figure 1. However, the inoculum size for these two strains was exactly the same in Figure 5, yet the number of lung cfu at Day 7 was greater in 602-infected mice as compared with those infected with CIP3-602. We also observed similar deposition but more rapid clearance of CIP3-602 as compared with 602 in one other experiment (not shown). In addition, the inflammatory reaction was greater in mice infected with 602 as compared with CIP3- 602 (see Figure 2), suggesting a requirement for increased cell recruitment by lung macrophages unable to contain the yeast. Finally, when both CD4 and CD8 T cells were depleted before infection, 602 increased in the lungs over the ensuing 42 d, whereas CIP3-602 did not. Indeed, both the 602- and TYCC38-602-infected lung cells secreted IFN- upon Con A stimulation ex vivo, whereas lung cells from CIP3-602-infected mice made significantly less. These data are compatible with the failure of CIP3-602-infected mice to mount any type of immune response arguing that the host responded to CIP3-602 as if it were relatively innocuous. These findings emphasize the importance of employing two acapsular control strains, 602 and CIP3-602, instead of 602 alone. For molecular genetic manipulations, such as complementation, it is essential to introduce plasmid DNA into cells. In cryptococcal strains where genetic crosses are possible, crossing with a wild-type yeast can eliminate plasmid DNA, an optimal scenario for testing the role of CAP64. Though a sexual cycle exists in C. neoformans, serotype D, the benefits of genetic crosses are not available for genetic manipulations with serotype A strains, due to the lack of MATa strains. Serotype A strains are most relevant to human disease and, therefore, a serotype A strain was studied here. Insertion of the transforming plasmid into the 602 chromosome cannot be controlled, and multiple copies of the plasmid DNA are present in both the CIP3-602 and TYCC38-602 (10). We believe that the presence of plasmid DNA reduced lung virulence in CIP3-602. We speculate this occurred, because the plasmid DNA interrupted a gene (or genes) and/or the rate of expression of a gene that facilitated growth in the host environment or regulated resistance against host defenses or both. What is clear is that introduction of the plasmid DNA neither resulted in the production of capsule nor in enhanced virulence, as was observed when the CAP 64 gene is inserted into 602 to yield TYCC38-602. Thus the fact that empty vector transformation decreased virulence makes the role of the inserted capsule gene even more impressive. It would be highly fortuitous that insertion of the DNA expressing a capsular gene would have simultaneously resulted in capsule expression and upregulation of another previously silent or suppressed virulence gene. Indeed, as indicated in MATERIALS and METHODS, there was no difference among the three strains in growth rate at 37°C nor in melanin production, two important determinants of virulence. Thus, there was no evidence to indicate that differences in virulence between 602 and CIP3-602 were due to growth rate or degrees of melanin production.

In summary, encapsulation of C. neoformans is alone sufficient to impart increased pulmonary virulence and the potential for dissemination to the brain. This observation provides an additional impetus to seek specific therapeutic agents that block the ability of C. neoformans to synthesize a capsule.