Avaliação do papel da IL-10 endógena na infecção pela bactéria Brucella abortus em modelo murino

Abstract

Brucella abortus is a Gram-negative pathogenic bacterium which causes a chronic disease in bovine, humans and others species called brucellosis. The host ability to mount a Th1/pro-inflammatory response against the bacteria is crucial to control and to solve the infection. It is suggested that IFN-γ produced by Th1cells is crucial to control the infection in mice. The interleukin IL-10 is considered to be responsible to decrease the IFN-γ production in vitro interfering directly in the pathogen elimination. During B. abortus infection, IL-10 acts limiting inflammatory response favoring the establishment persistence infection in mice. The goal of this study was evaluated the role of endogenous IL-10 during B. abortus infection. To assess the role of IL-10 in vivo, IL-10 knockout (IL-10 KO) mice or 129 Sv/Ev mice were infected with B. abortus strain S2308 and the number of viable bacteria recovery from the spleen were evaluated. One, 2, 3, 6 and 14 weeks post infection, IL-10 KO mice showed lower number of bacteria in the spleen when compared to wild type mice during all the time points. It was observed the increase of the difference at 14th week post infection. Additionally, it was demonstrated by survival curve in a period of 14 weeks post infection that approximately 35% of IL-10 KO mice died only at 2 or 3 weeks. Furthermore, the IFN-γ, IL-10, IL-17 and TNF-α production were measured in the supernatant from spleen cell culture in vitro when the cells were stimulated with alive B. abortus (S2308), heat killed B. abortus (HKBa), E. coli LPS or ConA. IL-10 KO cells showed greater increase in IFN-γ and TNF-α level in the supernatant from these cells when they were stimulated with Brucella when compared to wild type cells production. IL-17 was only detected in the supernatant from IL-10 KO cells in all time points analyzed. In addition, the TNF-α, IL-6, IL-12-p40, NO and IL-10 levels were evaluated in bone-marrow macrophage derived cell supernatant. TNF-α, NO, IL-6 and IL-12 showed augmented in IL-10 KO cells when stimulated with HKBa. However, only in supernatant from 129 Sv/Ev mice it was observed IL-10 production. In bone-marrow dendritic cell supernatant greater levels of TNF-α and IL-12-p40 were observed in IL-10 KO cells when compared to wild type mice cells during all time points analyzed when the cells were stimulated with all stimulus. The TGF-β expression was evaluated by real time PCR in splenic cells culture at 0 (non-infected cells), 1, 2 and 6 weeks after infection with S2308. At 2 weeks post infection the IL-10 KO cells demonstrated greater increased of TGF-β expression when compared to wild type cells. At 6 weeks post infection the TGF-β expression profile showed inverted. Additionally, the level of CD4+CD25+Foxp3+ (Treg) cells was assessed at spleen of infected mice by flow cytometry during the infection process. This population was increased at 3th and 6th weeks post infection in IL-10 KO mice. Descriptive histopathology analyses were done at liver level demonstrating a gradual diminishment of granuloma in both kinds of analyzed animals. However, the decrease pathology was more effective at IL-10 KO livers considering the granuloma area, measured by digital morphometry, and the hepatic parenchyma recovery. Taken together, the data provided by this work support that the lack of IL-10 is related to higher resistance to B. abortus infection in murine infection throughout the increase production of pro-inflammatory cytokines culminating with better bacteria elimination and a quicker tissue pathological resolution leading to a more effective control of this infection.