Dual Role for Nitric Oxide in Paracoccidioidomycosis: Essential for Resistance, but Overproduction Associated with Susceptibility 1 Fla ´ via R. F. Nascimento, Vera L. G. Calich, Dunia Rodrı ´guez, and Momtchilo Russo 2 Using a murine model of susceptibility and resistance to paracoccidioidomycosis, we have previously demonstrated that immu- nosuppression occurs in susceptible (B10.A), but not in resistant (A/Sn), mouse strains. Accumulating evidence shows that NO is involved in the induction of T cell immunosuppression during infection as well as in the killing of Paracoccidioides brasiliensis. In the present work, we focused on NO and other macrophage products that could be associated with resistance or susceptibility to paracoccidioidomycosis. A striking difference was related to NO and TNF production. Macrophages from B10.A mice produced high and persistent NO levels, while in A/Sn animals, TNF production predominated. In in vitro cultures, P. brasiliensis-infected macrophages from A/Sn mice also produced large amounts of TNF, while B10.A macrophages only produced NO. TNF production by B10.A macrophages appeared to be suppressed by NO, because the addition of aminoguanidine sulfate, an inducible NO synthase (NOS2) inhibitor, resulted in TNF production. These results suggested that enhanced TNF or NO production is associated with resistance and susceptibility, respectively. However, regardless of the mouse strain, NOS2-deficient or aminoguanidine sulfate-treated mice presented extensive tissue lesions with increased fungal load in lungs and liver compared with their controls. We conclude that NOS2-derived NO is essential for resistance to paracoccidioidomycosis, but overproduction is associated with susceptibility. The Journal of Immunology, 2002, 168: 4593– 4600. I nfection by the dimorphic fungus Paracoccidioides brasil- iensis (Pb) 3 can result in a systemic granulomatous disease designated paracoccidioidomycosis (Pb mycosis). Pb myco- sis presents a gamut of clinical forms ranging from a localized and benign disease to a relentlessly progressive and potentially lethal systemic infection (1). The outcome of the infection depends on several factors, such as the host responses and the virulence of the infecting agent (2, 3). We have previously characterized an isogenic murine model in which polar forms of the disease were reproduced. The B10.A mouse strain was found to be susceptible while the A/Sn strain was extremely resistant to Pb infection. Resistant mice are able to con- trol the infection, while susceptible animals develop a progressive disseminating disease with increased recovery of fungal cells from several organs (4). Although the mechanism(s) involved in resis- tance to Pb infection is poorly understood, it appears that macro- phages are fundamental for protection, as mice with blockade of macrophage functions by colloidal carbon are extremely suscepti- ble to infection (5). A key antimicrobial molecule involved in the killing of intra- cellular pathogens is NO, which is generated from the amino acid L-arginine by the inducible isoform of NO synthase (iNOS or NOS2). Although NO production is involved in Pb killing (6), it can also be involved in immunosuppression, because NO inhibits lymphoproliferation, TNF- production, and MHC class II mole- cule expression (7, 8). We have previously shown that in susceptible (B10.A), but not in resistant (A/Sn), mice, Pb infection is accompanied by suppres- sion of the Ab response to unrelated Ags and an impaired delayed- type hypersensitivity reaction (9, 10). In the present work, we se- lected these two polar mouse strains to ascertain by comparison which of the macrophage-derived products could be associated with resistance or susceptibility to Pb infection. We quantified the production of NO and other macrophage-derived metabolites, such as hydrogen peroxide and TNF-, in infected B10.A and A/Sn mice. We also determined the macrophage expression of MHC class II molecule (IA k ), as these two strains are identical in the IA region. In this study, we show that the major difference in macro- phage responses observed in these strains was related to NO and TNF- production. Macrophages from susceptible mice released high and persistent levels of NO throughout the infection, while macrophages from resistant mice presented persistent TNF- pro- duction. Incubation of resident macrophages from susceptible or resistant mice with Pb resulted in NO or TNF production, respec- tively. TNF- production by B10.A macrophages appeared to be suppressed by NO, because the addition of an NOS2 inhibitor, aminoguanidine hemisulfate (AG), restored TNF- production. These results indicate that NO might play a negative role in Pb infection. However, B6 mice with NOS2 deficiency were more susceptible to infection than NOS2-sufficient mice. Moreover, treatment with AG during the first 10 days of infection resulted in increased fungal load and more severe infection in both susceptible and resistant animals. Departamento de Imunologia, Instituto de Cie ˆncias Biome ´dicas, Universidade de Sao Paulo, Sao Paulo, Brazil Received for publication November 5, 2001. Accepted for publication February 20, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado de Sao Paulo, Coordenac ¸a ˜o de Apoio a ` Pesquisa e Ensino Superior, and Conselho Nacional de Pesquisas. 2 Address correspondence and reprint requests to Dr. Momtchilo Russo, Departa- mento de Imunologia, Instituto de Cie ˆncias Biome ´dicas IV, Universidade de Sao Paulo, Avenida Prof. Lineu Prestes 1730, CEP 05508-900, Sao Paulo SP, Brazil. E-mail address: momrusso@icb.usp.br 3 Abbreviations used in this paper: Pb, Paracoccidioides brasiliensis; AG, amino- guanidine; KO, knockout; NOS, NO synthase; p.i., postinfection. Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00