The role of nitric oxide (NO) in granulomas of Paracoccidioides brasiliensis-infected inducible NO synthase-deficient C57BL/6 mice (iNOS KO) and their wild-type counterparts and its association with osteopontin (OPN) and matrix metalloproteinases (MMPs) was studied. At 15 days after infection (DAI), iNOS KO mice showed compact and necrotic granulomas with OPN+ macrophages and multinucleated giant cells, whereas wild-type mice developed loose granulomas with many fungi and OPN+ cells distributed throughout the tissue. In addition, high OPN levels and fungal load were observed in iNOS KO mice. Both experimental groups had MMP-9 activity. At 120 DAI, iNOS KO had smaller granulomas with OPN+ cells, lower OPN levels, lower fungal load and decreased MMP-9 activity compared with wild-type mice. These findings suggest that NO has an important role in granuloma modulation, by controlling OPN and MMP production, as well as by inducing loose granulomas formation and fungal dissemination, resulting, at later phases, in progression of paracoccidioidomycosis.

The participation of osteopontin (OPN) in Paracoccidioides brasiliensis infected mice, its association to granulomatogenesis, severity of infection, pattern of lesions, nitric oxide (NO) levels and fungal load were evaluated in this investigation. Immunohistochemistry analysis showed marked OPN staining in extracellular matrix and in macrophages and multinucleated giant cells at the center of lesions, suggesting a possible role of OPN in the distribution of these cells within the granulomas. At 15 days post-infection with a virulent P. brasiliensis isolate, OPN+ cells were more numerous and intensely immunostained in the loose granulomas of susceptible mice than in those of resistant mice. In addition, high fungal loads and low NO levels were observed in susceptible mice. At 120 days after infection, resistant mice had increased total OPN levels (ELISA) and OPN positivity in compact granulomas, higher NO levels and lower fungal loads than susceptible mice. Residual lesions associated with low OPN levels, high NO and control of fungal dissemination were observed in both mouse strains at 120 days post-infection with the slightly virulent fungal isolate. Therefore, OPN could be associated with higher severity of the disease in an early phase of infection and with a degree of control of the progressive infection.

The important role of interferon-gamma (IFN-c) in protective immunity in mycosis is well established, except for its participation in fungal granulomas. Herein, we employ immunohistochemical reactions to describe the in situ localization of IFN-c in granulomas of susceptible (B10.A) and resistant (A/J) mice to infection with Paracoccidioides brasiliensis (Pb). After infection with the highly virulent Pb18, IFN-c-positive lymphomononuclear cells were localized mainly at the periphery of granulomas in both mouse strains. The numbers of positive cells found in compact granulomas of A/J mice increased significantly from 15 to 120 days postinfection. At this time, significantly more positive cells were detected in the compact granulomas of resistant mice than in the loose, multifocal lesions of the susceptible ones. In infection with the slightly virulent Pb265, the same pattern of IFN-c localization was found as in Pb18 infection, but there was decreased staining at 120 days due to the presence of only residual lesions in both mouse strains. The marked IFN-c staining observed in the granulomas of resistant mice at the later stage of Pb infection confirms its importance in fungal dissemination control, and suggests a contribution to the development of paracoccidioidal granuloma.

Isogenic mouse strains have previously been characterized as susceptible or resistant to Paracoccidioides brasiliensis infection; the former presented anergy in delayed-type hypersensitivity reactions (DTH) and progressive disease with high numbers of colony-forming units (CFU), while the later presented preserved DTH responses and control of the infectious process. Here, we studied whether susceptible mice infected with P. brasiliensis and treated with the antifungal drug trimethoprim-sulfamethoxazole (SXT) had their behavior pattern altered to the one observed in infected resistant mice. Therapy with either 30 or 150 mg SXT day(-1 )kg(-1), instituted 24 h after infection, elicited more adequate DTH responses than those of non-treated mice, and also diminished the number of viable fungi in the spleen and lungs, but not in epiploo and liver, indicating a partial control of the infectious process. This phenomenon was confirmed by histopathological analyses, in which the spleen was found to be the organ in which differences between the treated and non-treated groups were most remarkable. In control non-treated mice, the spleen parenchyma showed multiple granulomatous foci presenting giant cells, plasmocytes and many yeasts of P. brasiliensis with well-preserved morphology and abundant budding, whereas SXT-treated mice, independently of the dosage used, had no granulomas within the parenchyma and only few capsular lesions, mainly composed of pseudoxantomatous macrophages. Treatment with 150 mg day(-1 )kg(-1) (the dose considered to evoke best responses in CFU assays when therapy was instituted 24 h after infection), initiated at different times after infection, did not led to sustained DTH reactions, but provided an effective control of the disease when therapy began until the 15(th) day post infection, as showed by CFU assays. We conclude that reversal from the susceptible to the resistant pattern in experimental paracoccidioidomycosis can occur, but only when therapy with an adequate SXT dosage is instituted at a very initial phase of the infection. These protocols may constitute a model for further investigations concerning responses during antifungal therapy.

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