Differential regulation of immune responses by highly and weakly virulent Cryptococcus neoformans isolates

Abstract

Early inflammatory responses, delayed-type hypersensitivity (DTH) responses, and cytokine profiles were studied in mice infected by the pulmonary route with either a highly virulent isolate (NU-2) or a weakly virulent isolate (184A) of Cryptococcus neoformans. After infection, NU-2 remained in the lungs and the capsule became more pronounced during the first 24 h, whereas 184A induced an immediate inflammatory reaction and was rapidly cleared from the lungs. Cryptococcal antigen (GXM) appeared in sera early after infection with NU-2 and increased over the entire observation period. There was no detectable GXM in sera from 184A-infected mice. Both C. neoformans isolates induced anticryptococcal cell-mediated immune responses, but the responses had different profiles. DTH in NU-2-infected mice appeared at day 15 after infection and waned by day 21, whereas DTH in 184A-infected mice was present by day 5 and continued to increase. T helper 1 (Th1) cytokines (interleukin 2 [IL-2] and gamma interferon) were made by spleen cells early after infection with either isolate. NU-2-infected mice lost their ability to produce these cytokines, but 184A-infected mice retained it. IL-4, a Th2 cytokine, was not detected in infected mice. The regulatory cytokine IL-10 was made by spleen cells early but not later after infection with the highly virulent isolate and was not produced by spleen cells from 184A-infected mice. IL-10-deficient mice survived an NU-2 infection significantly longer than wild-type mice, suggesting that IL-10 is important in down-regulating the protective immune response. The induction of anergy appears to be responsible for the inability of NU-2-infected mice to control a C. neoformans infection.

FIG. 1

CFU cultured from the lungs of mice immediately or 24 h after intratracheal infection with C. neoformans NU-2 or 184A. ∗, statistically significant (P < 0.001) compared to NU-2-infected lungs 24 h after infection. The data presented represent the mean CFU ± standard errors of the means (error bars) for five animals/experimental group.

FIG. 2

Lung sections taken from mice 24 h after infection with C. neoformans NU-2 (A and B) or 184A (C and D). Sections were stained with hematoxylin and eosin (A and C) or mucicarmine (B and D). There were three mice in each experimental group. Photomicrographs are from one representative animal from each group. Encapsulated cryptococcal cells are indicated by the arrows. Magnification, ×200 (A and C) or ×400 (B and D).

FIG. 3

Lung section taken 24 h after infection with C. neoformans NU-2. Shown in both panels is normal lung tissue (lower left) adjacent to patchy inflammed tissue (central and lower right). Magnification, ×200 (A) or ×400 (B), with polymorphonuclear leukocytes indicated by the arrows.

FIG. 4

Serum GXM concentrations in mice infected with NU-2 and 184A. Data presented are means ± standard errors of the means (SEM) (error bars) for three to five individual mice tested at each time period.

FIG. 5

DTH footpad reaction to cryptococcal CneF antigen in CBA/J mice infected with C. neoformans NU-2 or 184A. Statistically significant (P = 0.0003) data compared to the DTH response of NU-2-infected mice at 28 to 29 days of infection (∗) and statistically significant (P = 0.02) data compared to the response of NU-2-infected mice at day 15 of infection (∗∗) are indicated. The data shown are combined from two separate experiments and represent the mean increases in paw thickness ± standard errors of the means (error bars) for three to nine animals/time period.

FIG. 6

IL-2 secreted by spleen cells taken from CBA/J mice at various times after infection with C. neoformans NU-2 or 184A. Spleen cells were cultured with or without the addition of cryptococcal antigen CneF, and supernatants were harvested 24 h after the initiation of culture. There was no difference in IL-2 levels between the medium alone and CneF-stimulated cultures when spleen cells were harvested from sham-treated mice; therefore, the data shown as dashed horizontal lines represent the variation (± standard errors of the means [SEM]) of the cytokine response of the sham-treated controls to medium alone and to CneF. ∗, statistically significant (P = 0.04) compared to IL-2 secretion from spleen cells of NU-2-infected mice at day 24 or 27 of infection. Vertical error bars represent the variation (±SEM). There were three to four animals in each experimental group, and the experiment was repeated once.

FIG. 7

IFN-γ secreted by spleen cells taken from CBA/J mice at various times after infection with C. neoformans NU-2 or 184A. Spleen cells were cultured with or without the addition of cryptococcal antigen CneF, and supernatants were harvested 48 h after the initiation of culture. There was a difference in the IFN-γ level between the medium alone and CneF-stimulated cultures when spleen cells were harvested from sham-treated mice; therefore, the data shown for the medium response (± standard errors of the means [SEM]) (error bars) are presented as the horizontal dotted lines, and the response to CneF (±SEM) is represented as horizontal dashed lines. Statistically significant (P = 0.049) data compared to IFN-γ secretion by spleen cells from NU-2-infected mice at day 24 after infection (∗) and statistically significant (P < 0.0002) data compared to the secretion of IFN-γ by spleen cells of NU-2-infected mice at day 27 or 30 after infection (∗∗) are indicated. Vertical error bars represent the variation (±SEM). There were five to six animals/experimental group, and the experiment was repeated once.

FIG. 8

IL-4 secreted by spleen cells taken from CBA/J mice at various times after infection with C. neoformans NU-2 or 184A. Spleens were cultured with or without the addition of cryptococcal antigen CneF, and supernatants were harvested 48 h after the initiation of culture. There was no difference in IL-4 levels between the medium alone and CneF-stimulated cultures when spleen cells were harvested from sham-treated mice; therefore, the data shown as dashed horizontal lines represent the variation (± standard errors of the means [SEM]) of the cytokine response of the sham-treated controls to medium alone and to CneF. Vertical error bars represent the variation (±SEM). There were five to six animals/experimental group, and the experiment was repeated once.

FIG. 9

IL-10 secreted by spleen cells taken from CBA/J mice at various times after infection with C. neoformans NU-2 or 184A. Spleen cells were cultured with or without the addition of cryptococcal antigen CneF, and supernatants were harvested 48 h after the initiation of culture. There was no difference in IL-10 levels between the medium alone and CneF-stimulated cultures when spleen cells were harvested from sham-treated mice; therefore, the data shown as dashed horizontal lines represent the variation (± standard errors of the means [SEM]) of the cytokine response of the sham-treated controls to medium alone and to CneF. Statistically significant (P = 0.04) data compared to IL-10 secretion from spleen cells of 184A-infected mice at day 15 after infection (∗) are indicated. Vertical error bars represent the variation (±SEM). There were three to four animals in each experimental group, and the experiment was repeated once.

FIG. 10

Survival of C57BL/6J or IL-10 knockout mice infected by the intratracheal route with 10⁵ C. neoformans NU-2 organisms. The survival rate of IL-10 knockout mice was significantly higher (P = 0.0002) than that of C57BL/6J mice. Data represent the survival rates for 19 animals/mouse strain.