Nitric oxide (NO), a key antimicrobial molecule, was previously shown to exert a dual role in paracoccidioidomycosis, an endemic fungal infection in Latin America. In the intravenous and peritoneal models of infection, NO production was associated with efficient fungal clearance but also with non-organized granulomatous lesions. Because paracoccidioidomycosis is a pulmonary infection, we aimed to characterize the role of NO in a pulmonary model of infection.
C57Bl/6 wild type (WT) and iNOS−/− mice were i.t. infected with 1×106
Paracoccidioides brasiliensis yeasts and studied at several post-infection periods. Unexpectedly, at week 2 of infection, iNOS−/− mice showed decreased pulmonary fungal burdens associated with an M2-like macrophage profile, which expressed high levels of TGF-β impaired ability of ingesting fungal cells. This early decreased fungal loads were concomitant with increased DTH reactions, enhanced TNF-α synthesis and intense migration of activated macrophages, CD4+ and CD8+ T cells into the lungs. By week 10, iNOS−/− mice showed increased fungal burdens circumscribed, however, by compact granulomas containing elevated numbers of activated CD4+ T cells. Importantly, the enhanced immunological reactivity of iNOS−/− mice resulted in decreased mortality rates. In both mouse strains, depletion of TNF-α led to non-organized lesions and excessive influx of inflammatory cells into the lungs, but only the iNOS−/− mice showed increased mortality rates. In addition, depletion of CD8+ cells abolished the increased migration of inflammatory cells and decreased the number of TNF-α and IFN-γ CD4+ and CD8+ T cells into the lungs of iNOS−/− mice.
Our study demonstrated that NO plays a deleterious role in pulmonary paracoccidioidomycosis due to its suppressive action on TNF-α production, T cell immunity and organization of lesions resulting in precocious mortality of mice. It was also revealed that uncontrolled fungal growth can be overcome by an efficient immune response.
Paracoccidiodomycosis is a human systemic mycosis endemic in Latin America that has a wide spectrum of manifestations ranging from localized to fatal disseminated forms. Both in humans and experimental models, immunoprotection is mediated by T cell immunity whereas immunosuppression is associated with the severe forms of the disease. The literature shows that nitric oxide (NO) produced by the enzyme nitric oxide synthase-2 (NOS2 or iNOS) is the major fungicidal component of phagocytic cells. The role of NO production was previously investigated in the intra-peritoneal and intravenous murine models of Paracoccidioides brasiliensis infection. The human paracoccidioidomycosis is believed to be acquired by the respiratory route, thus our study aimed to characterize the role of NO production in a pulmonary model of infection. We verified that, paradoxically, absence of NO production by iNOS- deficient mice resulted in less severe disease and increased survival times. This was associated with increased development of cellular immunity and enhanced synthesis of TNF-α which enhances cell migration to the site of infection and contributes to the better organization of lesions. Our work highlighted the deleterious effect of excessive NO production in pulmonary paracoccidioidomycosis, and demonstrated that uncontrolled fungal growth can be overridden by an efficient immune response.