Interleukin-12 is essential for a protective Th1 response in mice infected with Cryptococcus neoformans

Abstract

To analyze the roles of interleukin-12 (IL-12) and the IL-12-dependent Th1 response in resistance to Cryptococcus neoformans, we have established a chronic infection model in wild-type mice and in mice with targeted disruptions of the genes for the IL-12p35 and IL-12p40 subunits (IL-12p35(-/-) and IL-12p40(-/-) mice, respectively) as well as in mice with a targeted disruption of the IL-4 gene. Long-term application of exogenous IL-12 prevented death of infected wild-type mice for the entire period of the experiment (up to 180 days) but did not resolve the infection. Infected IL-12p35(-/-) and IL-12p40(-/-) mice died significantly earlier than infected wild-type mice, whereas infection of IL-4-deficient mice led to prolonged survival. Interestingly, infected IL-12p40(-/-) mice died earlier and developed higher organ burdens than IL-12p35(-/-) mice, which, for the first time in an infection model, suggests a protective role of the IL-12p40 subunit independent of the IL-12 heterodimer. The fungal organ burdens of IL-4-deficient mice and IL-12-treated wild-type mice were significantly reduced compared to those of untreated wild-type mice and IL-12-deficient mice. Histopathological analysis revealed reduction of the number of granulomatous lesions following treatment with IL-12. Susceptibility of both IL-12p35(-/-) and IL-12p40(-/-) mice was associated with marginal production of gamma interferon and elevated levels of IL-4 from CD4(+) T cells, which indicates Th2 polarization in the absence of IL-12, whereas wild-type mice developed a Th1 response. Taken together, our data emphasize the essential role of IL-12 for protective Th1 responses against C. neoformans.

FIG. 1

Effect of IL-12 treatment on survival of mice infected with C. neoformans. Mice were infected by i.v. injection with 10⁴ CFU of C. neoformans and treated intraperitoneally daily during the first week of infection with either buffer or IL-12 (0.25 μg/mouse) beginning 1 day before infection. Treatment was continued twice weekly with 0.1 μg/mouse for the long-term treatment group. Seven to ten mice per group were monitored. At day 134 after infection, the long-term treatment group was divided. For five mice, treatment with IL-12 was continued, while for the other five mice, treatment was terminated. The mice receiving continuous treatment with IL-12 survived until day 180, the end of the experiment (not shown). The figure represents the results of one typical experiment of six similar experiments.

FIG. 2

Fungal burden in lungs of mice 21 days after infection with C. neoformans. Mice were infected by i.v. injection with 10⁴ CFU of C. neoformans. For the group of IL-12-treated wild-type mice, daily treatment with IL-12 (0.25 μg/mouse intraperitoneally) was started 1 day before infection and continued for the first week. Treatment was continued twice weekly with 0.1 μg/mouse for the next 2 weeks. Twenty-one days after infection, the mice were killed by CO₂ asphyxiation and organs were homogenized. Serial dilutions of the homogenates were plated on Sabouraud agar, and colonies were counted after 48 h of incubation at 30°C. Each point is the value for one mouse, and the median values are indicated by the short horizontal lines. Values shown in the figure are adjusted to the whole organ. This figure shows the results of one representative experiment of 12 similar experiments.

FIG. 3

Histopathological analysis of livers from IL-12-treated or untreated mice 21 days after infection with C. neoformans. Liver sections of wild-type mice (A, B, and C), IL-12p35^−/− mice (D), and IL-12p40^−/− mice (E) 21 days after the infection with C. neoformans are shown. The livers were fixed in 10% phosphate-buffered formalin. Tissues were embedded in paraffin, and sections 5 μm thick were stained with hematoxylin and eosine. Microphotographs A and B demonstrate representative liver sections from animals treated with either IL-12 (B) or buffer (A) for 3 weeks. The bars in panels A and B represent 0.3 mm, while the bars in panels C, D, and E represent 0.05 mm. Photographs C, D, and E demonstrate individual lesions typically found. A representative granuloma with phagocytosed cryptococci in epitheloid giant cells is seen in wild-type mice (C) and IL-12p35^−/− mice (D). In IL-12p40^−/− mice (E), primarily “cystic” lesions can be found. The figure shows two of five experiments analyzed histologically.

FIG. 4

Number of granulomatous lesions in livers from IL-12-treated or untreated mice 21 days after infection with C. neoformans. Mice were infected with 10⁴ CFU of C. neoformans and treated with IL-12 for 3 weeks as described in the legend to Fig. 2. The number of granulomatous lesions in the liver from infected mice was determined by counting the mean number of granulomatous lesions in 20 randomized areas of 0.08 mm². The mean value and standard deviation for three animals per group is shown. The differences between values with and without IL-12 treatment were found to be statistically significant by the two-tailed Student’s t test (P = 0.001 [∗]; P = 0.002 [∗∗]; P = 0.014 [∗∗∗].

FIG. 5

Net cytokine secretion of total spleen cells from mice infected with C. neoformans. Spleens were taken from three mice per group 21 days after infection with 10⁴ CFU of C. neoformans. Spleen cells were depleted of CD4⁺ or CD8⁺ T cells by using specific antibodies and magnetic beads. As assessed by subsequent fluorescence-activated cell sorter analysis, depletion of CD4⁺ or CD8⁺ T cells was complete (100%). The graph shows cytokine secretion after specific stimulation with heat-killed cryptococci with background values (medium controls) subtracted. The levels of IFN-γ and IL-4 in splenocyte cultures incubated with medium for 48 h were less than 5 and 10%, respectively. Polyclonal stimulation with anti-CD3 was similar between different groups (not shown). Levels of IFN-γ and IL-4 were quantified in 48-h culture supernatants by an enzyme-linked immunosorbent assay. Since the concentration of cells was kept constant at 2.5 × 10⁶ cells/ml in total and depleted samples, bars representing the CD8⁺-depleted samples reflect CD4⁺-enriched cultures with elevated cytokine production. This figure demonstrates the results of one representative experiment of nine similar experiments with cytokine analyses of lymphocytes cultured ex vivo.