The gamma interferon receptor is required for the protective pulmonary inflammatory response to Cryptococcus neoformans

Abstract

Mice with a null deletion mutation in the gamma interferon (IFN-gamma) receptor gene were used to study the role of IFN-gamma responsiveness during experimental pulmonary cryptococcosis. Cryptococcus neoformans was inoculated intratracheally into mice lacking the IFN-gamma receptor gene (IFN-gammaR-/-) and into control mice (IFN-gammaR+/+). The numbers of CFU in lung, spleen, and brain were determined to assess clearance; cytokines produced by lung leukocytes were measured, and survival curves were generated. In the present study, we demonstrate the following points. (i) IFN-gammaR-/- mice are markedly more susceptible to C. neoformans infection than IFN-gammaR+/+ mice. (ii) In the absence of IFN-gamma signaling, pulmonary CFU continue to increase over the course of infection, and the infection disseminates to the brain. (iii) In the absence of IFN-gamma receptor, recruitment of inflammatory cells in response to pulmonary cryptococcal infection is not impaired. (iv) At week 5 postinfection, IFN-gammaR-/- mice have recruited greater numbers of leukocytes into their lungs, with neutrophils, eosinophils, and lymphocytes accounting for this cellular increase. (v) IFN-gamma signaling is required for the development of a T1 over a T2 immune response in the lung following cryptococcal infection. These results indicate that in the absence of IFN- gamma responsiveness, even though the recruitment of pulmonary inflammatory cells is not impaired and the secretion of IFN-gamma is not affected, IFN-gammaR-/- mice do not have the ability to resolve the cryptococcal infection. In conclusion, our data suggest that proper functional IFN-gamma signaling, possibly through a mechanism which inhibits the potentially disease-promoting T2 response, is required for mice to confine the cryptococcal infection.

FIG. 1.

(A) Effect of IFN-γ receptor deletion on the survival of mice infected with C. neoformans. Ten mice from each group of IFN-γR^+/+ and IFN-γR^−/− mice were intratracheally inoculated with 10⁴ CFU of C. neoformans 52D. Survival experiments were carried out to day 49. The difference in survival at day 49 between IFN-γR^+/+ and IFN-γR^−/− mice was significant; P < 0.005. (B) Effect of IFN-γ receptor deletion on pulmonary growth and dissemination of C. neoformans in infected IFN-γR^+/+ and IFN-γR^−/− mice. Mice were inoculated intratracheally with 10⁴ CFU of C. neoformans 52D and assayed at weeks 2 and 5 postinoculation. Total CFU per organ were determined, and numbers in each column indicate the number of mice which had a C. neoformans burden in that organ. All mice had detectable CFU in the lungs. #, P < 0.05, and *, P < 0.001, compared to those for wild-type mice at the same time points; n = 12 for each group (two separate experiments of four and eight each). Values are given as means ± SEM.

FIG. 2.

Lung pathology in IFN-γR^+/+ and IFN-γR^−/− mice infected with C. neoformans at 5 weeks postinfection. (A) Photomicrograph of hematoxylin-and-eosin-stained sections from the lung of a C. neoformans-infected IFN-γR^+/+ mouse (magnification, ×66). Note only the localized area of leukocyte infiltrate. (B) Photomicrograph of hematoxylin-and-eosin-stained sections from the lung of a C. neoformans-infected IFN-γR^−/− mouse (magnification, ×66). Note the widespread leukocyte infiltrates surrounding numerous encapsulated cryptococci. (C) High-powered examination of an inflammatory focus in an IFN-γR^+/+ mouse lung showing the leukocyte infiltrate with predominantly mononuclear cells and no obvious cryptococci (magnification, ×132). (D) High-powered examination of an inflammatory focus in IFN-γR^−/− mouse lung showing massive infiltration of eosinophils (magnification, ×132). (E. and F) Representative lung sections were stained with Masson trichrome for collagen and other matrix proteins showing that the IFN-γR^−/− mouse (panel F) had more intense deposition than the IFN-γR^+/+ mouse (panel E) (magnification, ×66).

FIG. 3.

Production of MIP-2 and KC chemokines by lung leukocytes from C. neoformans-infected IFN-γR^+/+ and IFN-γR^−/− mice. At week 3 and week 4 postinoculation, total lung leukocytes were isolated and cultured at 5 × 10⁶ cells/ml with heat-killed strain 52D for 24 h. Culture supernatants were analyzed by ELISA for chemokines as described in Materials and Methods. #, P < 0.05, and *, P < 0.001, compared to those for wild-type mice at the same time points; n = 9 per data point, pooled from two separate experiments. Values are given as means ± SEM.

FIG. 4.

(A) Cryptococcal burdens of lung macrophages from C. neoformans-infected IFN-γR^+/+ and IFN-γR^−/− mice. At week 4 postinoculation, total lung leukocytes were isolated and cultured in 60-mm-diameter petri dishes at 5 × 10⁶ cells/ml. After 24 h, plates were washed three times with HBSS to remove nonadherent cells. Adherent lung macrophages were isolated with trypsin-EDTA and then cultured at 10⁵ cells/well in 96-well plates. CFU were determined as described above at 0 and 24 h after culture. *, P < 0.001, compared to cultures from wild-type mice at the same time point; #, P < 0.001, compared to cultures from the same mice at the different time point; n = 8 per data point, pooled from two separate experiments. Values are given as means ± SEM. (B) Photomicrographs of lung macrophages cultured in vitro for 24 h. Cryptococcal foci (shown with arrows) grew in cultures of lung macrophages from IFN-γR^−/− mice, but not those from IFN-γR^+/+ mice.

FIG. 5.

Production of T1- and T2-type cytokines by lung leukocytes from C. neoformans-infected IFN-γR^+/+ and IFN-γR^−/− mice. At week 5 postinoculation, total lung leukocytes were isolated and cultured at 5 × 10⁶ cells/ml for 24 h in vitro with or without heat-killed strain 52D. Culture supernatants were analyzed by ELISA for cytokines as described in Materials and Methods. #, P < 0.05, and *, P < 0.005, compared to those for wild-type mice at the same time points; n = 9 or 12 per data point, pooled from four separate experiments. Values are given as means ± SEM.