We utilized our previously-established protocol to induce chronic infection 3. Briefly, male Fischer 344 rats weighing 200–250 grams (6–8 weeks of age) were obtained form Taconic Farms (Germantown, NY). Rats were infected intratracheally or intraperitoneally with either 1 × 10⁷yeast or PBS as a control. Studies were also done with Brown Norway and Sprague Dawley rats. In some experiments, C. neoformans was killed in 70°C for 30 minutes prior to inoculation. All animal work was carried out with the approval of the Animal Use Committee at the Albert Einstein College of Medicine.

Based on previous experiments, C. neoformans, American Type Culture Collection (ATCC) strain 24067 was used to establish infection 8. In addition, C. albicans ATCC strain 200498 was used in certain experiments. Yeasts were grown in Sabouraud's broth at 30°C for 2 days, washed with PBS and counted using a hemacytometer prior to inoculation.

At different times following inoculation, rats were killed by inhalational exposure to CO₂. Tracheas were cannulated with a 16 G angiocath and lungs lavaged with 3 ml of PBS four times. Following BAL, the right lung was removed, homogenized in 5 ml of PBS, centrifuged and frozen in -80°C until analysis.

Lung homogenates and BAL fluid (BALF) were inoculated on Sabouraud's agar plates and incubated at 37°C for 72 hours. Colonies were then enumerated. Fungal burdens were log₁₀ converted and averaged. For the lungs, fungal burden was expressed per lung. For BAL, fungal burden was expressed per ml.

Chitinase activity assay was performed utilizing an established protocol 5. Briefly, BALF and whole lung homogenates were incubated with 4-methylumbelliferyl-D-N,N'-diacetylchitobioside (Sigma), which is cleaved by bioactive chitinase, thereby releasing a quantifiable fluorogenic substance, 4-methylumbelliferone (excitation 350 nm; emission 450 nm). Bioactive chitinase from Serratia marcescens (Sigma) was used for standard measurements. In some experiments, chitinase activity in the supernatant of C. neoformans cultures was measured.

BALF and lung homogenates from animals were pooled by infection status and time of infection. Ten microliters of the combined samples were separated with a 10% polyacrylamide gels under denaturing conditions and transferred to nitrocellulose. Western blots were performed with a rabbit polyclonal antibody against AMCase (1:500, a generous gift from Dr. Jack Elias, Yale University) and GAPDH (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA). The secondary antibody for both primary antibodies was a peroxidase-labeled goat anti-rabbit IgG (Southern Biotechnology Associates, Birmingham, AL) at a dilution of 1: 2500 for GAPDH detection and 1:5000 for AMCase detection. Bands were visualized using enhanced chemiluminescense according to the manufacturer's instructions (SuperSignal, Pierce Chemicals, Rockford, IL). Reactivity at 50 kDa, the approximate molecular weight of AMCase, was used to indicate AMCase expression 9.

Densitometry measurements of western blots were obtained with Image J software (NIH), and values were normalized to GAPDH. Results were reported in arbitrary units.

Lungs were fixed in formalin, embedded in paraffin, and subsequently processed for immunohistochemical analysis as described previously 10. Briefly, 5 μm sections were deparaffinized in xylene and rehydrated in graded concentrations of ethanol. Antigen retrieval was performed in a pressure cooker using 6.5 mM sodium citrate (pH 6.0). Sections were incubated with rabbit polyclonal antibody directed against AMCase (1:100, Dr. Jack Elias, Yale). Staining was visualized utilizing a commercially available kit (Santa Cruz). Negative controls were stained simultaneously with omission of primary antibody.

Student's T-test was used to compare individual differences in chitinase activity between infected and control animals. For multiple comparisons, one-way analysis of variance testing was used. Post-hoc testing was done using the Dunnett test. Differences between groups were considered significant at p < 0.05.

All rats inoculated with C. neoformans and C. albicans were actively infected at the time of chitinase determinations. Two days following intratacheal inoculation average log₁₀ fungal burdens in BAL (per ml) and lung (per entire right lung) were 4.42 ± 0.01 and 6.48 ± 0.01, respectively Fourteen days following intratracheal inoculation the average log₁₀ fungal burdens in BAL and lung were as follows: 3.97 ± 0.19 and 6.33 ± 0.03. At 2 months, the average lung fungal burden was log₁₀ 5.4 ± 0.02 (BAL was not tested). At 2 days following intraperitoneal inoculation the average log₁₀ fungal burden in the lung was 4.5 ± 0.04, however the fungal burden in the BAL was below the limits of detection (log₁₀ 2). For rats intratracheally infected with Candida albicans the log₁₀ fungal burden lung was 4.39 ± 0.06. No fungus was detected in the BAL limits of detection log₁₀ 2. Fungus was not detected in rats inoculated with heat-killed organisms.

As demonstrated in Figure 1A, a >2-fold increase in BALF chitinase activity was present as early as 2 days after intratracheal inoculation of C. neoformans. Similar chitinase activity was also present in the BALF of rats inoculated with C. albicans. Although chitinase activity appeared to be increased in the BALF of rats inoculated with heat killed C. neoformans on day 2, this increase was not statistically significant. Intraperitoneal inoculation of C. neoformans did not result in increased chitinase activity in BALF. No differences in chitinase activity were detected in whole lung homogenates on day 2 (Figure 1B).

Interestingly, 14 days after single intratracheal inoculation with C. neoformans, both BALF and whole lung chitinase activity from infected animals increased to >6-fold compared to controls (Figure 1B). In contrast, chitinase activity was not detected in either BALF or lung homogenates 14 days after inoculation with heat-killed C. neoformans. At 2-months of infection, chitinase activity remained elevated in BALF (3.5-fold) and whole lung homogenates (>2-fold) relative to PBS-inoculated animals (Figure 2). To insure that the observed chitinase activity was not due to fungal expression of chitinase, we tested cryptococcal culture supernatatants at multiple times following inoculation (days 1, 3, and 7). The density of organisms in culture by day 3 was approximately log₁₀ 7.30 ± 0.04 per ml. No chitinase activity was detected.

We repeated experiments with Brown Norway and Sprague Dawley rats because of their known tendencies to exhibit different degrees of TH2 polarization and allergic inflammation in response to antigenic stimulation 1112. At 2 weeks, chitinase activity was increased in the both the BAL and lung homogenate of infected rats relative to PBS-inoculated animals, regardless of rat strain (Table 1).

Because we observed significant increases in generalized chitinase activity in both BALF and whole lung from infected rats, and since several chitinase family members exhibit enzymatic activity, we sought to determine whether the specific expression of AMCase was induced by C. neoformans infection. As demonstrated in Figure 3, the pattern of AMCase expression paralleled chitinase activity. Specifically, increased AMCase expression was elevated in BALF as early as 2 days following intratracheal inoculation with both live C. neoformans (1.8-fold) and C. albicans (1.8-fold) compared with controls. There was no observable difference in AMCase expression with systemic infection. Also consistent with chitinase activity data, the increased AMCase expression in BALF persisted at day 14 (4-fold increase). Lastly, AMCase expression was not detected in lung homogenates at Day 2, but was clearly increased at day 14 compared to controls (7.3-fold corrected for GAPDH, data not shown).

As demonstrated in Figure 4, control animals demonstrated little signal in airway epithelium. In comparison, AMCase expression was seen at day 14 and at 2-months in airway epithelium (black arrows) and alveolar macrophages (white arrows).