IL-12P40 AND IL-18 PLAY PIVOTAL ROLES IN ORCHESTRATING INTERFERON-GAMMA-INDUCING NITRIC OXIDE PRODUCTION: THE MAJOR IMPORTANCE OF THIS PATHWAY IN THE CONTROL OF PRIMARY DENGUE VIRUS INFECTION IN MICE.

Vivian Vasconcelos Costa (UFMG); Caio Tavares Fagundes (UFMG); Deborah Fernandes Valadão (UFMG); Andréia Grossi Santos (UFMG); Thais Queiroz Morcatty (UFMG); Flávio Almeida Amaral (UFMG); Daniel Cisalpino (UFMG); Tarcília Aparecida da Silva (UFMG); Lirlândia Pirez de Sousa (UFMG); Kátia Daniela da Silveira (UFMG); Fernanda Matos Coelho (UFMG); Talles Prosperi de Paula (UFMG); Cristiano Xavier Lima (UFMG); Danielle da Glória de Souza (UFMG); Mauro Martins Teixeira (UFMG)

Introduction: Dengue is a mosquito-borne infection which has become a major international public health concern. Our group has recently developed murine models of infection for dengue virus (DENV) serotypes 2 and 3, which resembles the severe dengue human infection. Recently, an important role is attributed to cytokines in host response to DENV infections. Although, a strong cell-mediated immune response is critical for controlling viral infections and is regulated by a number of cytokines, including IL-12,  IL-18 and IFN-ã. Objectives: The aim of this study was to evaluate the role played by the IL-12p40, IL-18 and IFN-ã inducing- nitric oxide pathway in response to primary dengue virus infection. Methods:  Wild type mice (C57BL/6), deficient mice to IL-12p40 subunit (Il-12p40^‑/-), IL-18 (IL-18^-/-), IFN-ã (IFN-ã ^-/-) and iNOS (iNOS^-/-) were infected with the adapted DENV-3 (10 PFU/animal) by the i.p. route. After infection, lethality of animals was accompanied. Evaluation of clinical signals (hematocrit, platelets, plasmatic albumin), hepatic transaminases levels, mechanical hypernociception, production of inflammatory mediators (cytokines/ chemokines (ELISA) and neutrophils (MPO assay) on spleen, liver and lungs were made in the 5^th and in the 7^th day after infection. Tissue damage was verified by histological analyses. Expression of iNOS was evaluated by Immunohistochemical detection of iNOS in liver. Production of NO was evaluated in DCs culture (Griess method), and viral load in target organs was quantified by plaque assay in permissive cells (LLCMK2 cells). All procedures have been approved by ethics committee (CETEA/UFMG: protocol 113/09). Results: WT mice were able to produce IFN-ã after infection. This production was dependent of IL-12 and Il-18 cytokines. Furthermore, IL-12p40^-/-, IL-18^-/- and IFN-ã^-/- mice presented significantly higher lethality after infection when compared to WT mice. This reduced survival rate was associated with more severe disease manifestation when compared to control littermates. Hence, all deficient mice presented increased hemoconcetration, thrombocytopenia and production of pro-inflammatory mediators. This enhanced susceptibility to infection was characterized by higher tissue damage and reduced control of viral replication after infection of all deficient mice. Our results also showed that IFN-ã^-/- mice presented drastic reduction in NO production after DENV-3 infection. Interestingly, iNOS^-/- mice also presented enhanced lethality, a more severe disease development and increased viral loads after infection. Conclusion: Here, we conclude that IL-12p40 and IL-18 play pivotal roles in orchestrating interferon-gamma-inducing nitric oxide production and this is a fundamental pathway after DENV infection. In absence of these molecules, there is reduced ability to control viral replication by the host, resulting in a more severe disease manifestation. Support: CNPq, CAPES and FAPEMIG.