Persistent Cryptococcus neoformans pulmonary infection in the rat is associated with intracellular parasitism, decreased inducible nitric oxide synthase expression, and altered antibody responsiveness to cryptococcal polysaccharide

Abstract

Fungal pathogens are notorious for causing chronic and latent infections, but the mechanism by which they evade the immune response is poorly understood. A major limitation in the study of chronic fungal infection has been the lack of suitable animal models where the infection is controlled and yet persists. Pulmonary Cryptococcus neoformans infection in rats results in a diffuse pneumonitis that resolves without dissemination or scarring except for the persistence of interstitial and subpleural granulomas that harbor viable cryptococci inside macrophages and epithelioid cells. Infected rats are asymptomatic but remain infected for as long as 18 months after inoculation with C. neoformans. Containment of infection is associated with granuloma formation that can be partially abrogated by glucocorticoid administration. Using this model, we identified several features associated with persistent infection in the rat lung, including (i) localization of C. neoformans to discrete, well-organized granulomas; (ii) intracellular persistence of C. neoformans within macrophages and epithelioid cells; (iii) reduced inducible nitric oxide synthase expression by granulomas harboring C. neoformans; and (iv) reduced antibody responses to cryptococcal polysaccharide. The results show that maintenance of persistent infection is associated with downregulation of both cellular and humoral immune responses.

FIG. 1

Histopathology of persistent infection. Peribronchiolar infection (A) (magnification, ×82.5), subpleural infection (B) (magnification, ×82.5), and interstitial infection (C) (magnification, ×825). (D) Vacuolated macrophages some of which contain C. neoformans (arrow) (magnification, ×825) are also shown. C. neoformans and associated inflammatory cells disrupt the respiratory epithelium (6 months). (E) C. neoformans and cellular debris are present in the bronchiolar lumen which is lined by hypertrophic respiratory epithelium (magnification, ×165). (F) An interstitial granuloma at 18 months of infection can be seen. Arrows point to intracellular C. neoformans (mucin staining; magnification, ×825). (G) Large cluster of extracellular C. neoformans cells with minimal inflammation in the lung of a rat that received dexamethasone 1 week after infection (mucin staining; magnification, ×165). (H) Macrophages containing many C. neoformans organisms in the lung of the same rat (magnification, ×825). (I) In rats treated with dexamethasone at 11.5 months after infection, both intracellular (arrow) and extracellular (arrowhead) C. neoformans organisms are present (mucin staining; magnification, ×330). All staining was done with hematoxylin and eosin unless stated otherwise.

FIG. 2

NOS2 expression in persistent pulmonary infection. The graph demonstrates lung fungal burden (mean CFU/gram of tissue) at 0.5, 0.8, 1.5, 6, 12.5, and 18 months after infection. Results for rats at 0.5 and 0.8 months after infection were previously described and are presented here for comparison (12). These numbers were plotted in the same graph though they originated from a different experiment because the CFUs are very consistent in this system. Representative NOS2 staining (brown) within granulomas at 0.5 (A), 1.5 (B), 6 (C), and 12.5 (D) months are shown in the panels above. All magnifications, ×70. The lung sections from 0.5 and 0.8 months of infection were obtained from a previous experiment and were used as a positive control for these experiments (12).

FIG. 3

Double staining for NOS2 (blue) and GXM (brown). (A) Small interstitial granulomas (1.5 months after infection) containing GXM demonstrate NOS2 staining, whereas areas containing large amounts of GXM demonstrate little or no NOS2 staining. The inset in panel A shows interstitial granuloma at high magnification. (B) Area of confluent inflammation containing large amounts of GXM show small foci of NOS2 reactivity (1.5 months after infection). (C) Granulomas at 6 months contain GXM but demonstrate minimal NOS2 staining. All magnifications, ×175.

FIG. 4

GXM and tetanus toxoid antibody titers. Mean antibody titers (1/log₁₀ ± 1 standard deviation [SD]) to GXM (A) and tetanus toxoid (B) in uninfected and infected rats after various doses of vaccine (GXM-TT and dT). The P value for GXM titers for infected versus uninfected rats receiving GXM-TT after 3 doses of vaccine was 0.025. After three doses of tetanus toxoid, at least one rat in each group had a titer greater than the maximal limit detectable by the ELISA, so the SD was not calculated. The dotted line represents the minimal limit of detection.