Heat-stress-modulated induction of NF-κB leads to brucellacidal pro-inflammatory defense against Brucella abortus infection in murine macrophages and in a mouse model

Abstract

Background: Brucella causes a chronic and debilitating infection that leads to great economic losses and a public health burden. In this study, we demonstrated the brucellacidal effect of heat shock mediated by the induction of pro-inflammatory cytokines, reactive oxygen species (ROS) accumulation and apoptosis in murine macrophages and in mice.

Results: RAW264.7 cells were incubated at 43 °C, and BALB/c mice were subjected to whole body hyperthermia. The data showed a reduction in bacterial survival in the mice after daily heat exposure. This was accompanied by increased levels of cytokines TNF, IL-6, IL-1β and IFN-γ in the sera of the mice. Gene expression of NF-κB and inducible nitric oxide production were also induced in the mouse splenic cells. In parallel with the bacterial reduction in the mouse model, an increased bactericidal effect was observed in RAW264.7 cells after exposure to heat stress. In addition, the heat stress increased both the nuclear translocation of NF-κB and the expression of the heat shock proteins HSP70 and HSP90 in murine macrophages. Furthermore, heat exposure induced the increase of pro-inflammatory cytokines, ROS accumulation and apoptosis but did not affect the production of nitric oxide (NO) in macrophages.

Conclusion: This study demonstrated the induction of innate immune responses by heat stress that significantly reduced the intracellular survival of B. abortus in vitro and in vivo. Transcriptional factor NF-κB, which is a master regulator, could be termed a key activator of heat-induced immunity against Brucella. The increase in the expression and activation of NF-κB in splenic cells and macrophages was followed by enhanced antimicrobial effectors, including cytokines, ROS and NO that may contribute to the reduction of bacterial survival.

Keywords: B. abortus; Heat stress; Macrophage; NF-κB; ROS.

Fig. 1

Daily heat exposure restricted bacterial survival in mice. The bacterial burdens in the spleen and liver (a), weight of spleen (b) and liver (c) were evaluated two weeks post-infection in heat-exposed and non-exposed mice. Data represent the mean ± SD of replicates of six samples. Asterisks indicate significant differences (p < 0.05)

Fig. 2

Heat stress induced cytokine secretion. Expression of cytokines Il6, Il1b, Tnf, Mcp1, Ifng and Il10 by mouse splenic cells with and without exposure to heat quantified by RT-PCR (a). Quantification of cytokines: TNF (b), IL-6 (c), IL-1β (d), MCP-1 (e), and IFN-γ (f) in the sera of mice by flow cytometer and sandwich ELISA. Data represent the mean ± SD of replicates of six samples. Asterisks indicate significant differences (p < 0.05)

Fig. 3

Heat stress caused an increase in NF- κB and iNOS expression in splenic cells. The expression of heterodimers of NF- κB: p50, p52 and p65 from splenic cells was quantified by RT-PCR (a). Immunoblot analysis of the expression of heterodimers of NFkB: p50, p52 and p65 from splenic cells (b). Immunoblot analysis of iNOS expression following heat exposure (c). Asterisks indicate significant differences (p < 0.05)

Fig. 4

Bacterial killing activity was evaluated in murine macrophages exposed to heat stress. Bacterial internalization of B. abortus after treatment with heat stress (a). Intracellular growth of B. abortus in RAW 264.7 cells initially infected and exposed to heat (b). Intracellular growth of B. abortus in BMM initially infected and exposed to heat (c). Data represent the mean ± SD of triplicate experiments. Asterisks indicate significant differences (p < 0.01)

Fig. 5

Heat stress accelerated heat shock protein expression and NF- κB nuclear translocation but not NO production at 24 h pi. The heat shock response induced production of heat shock proteins (HSPs) (a), NF- κB p65 translocation (b), and immunoblot analysis of cytoplasmic and nuclear NF- κB p65 following heat exposure (c). Effect of heat stress on nitrite production as quantified by the Griess assay (d). Data represent the mean ± SD of triplicate experiments. (Scale bar = 5 μm)

Fig. 6

The effect of heat stress on the production of pro-inflammatory cytokines, ROS accumulation and apoptosis. The cells were infected with B. abortus and subjected to heat exposure. The production of pro-inflammatory cytokines and ROS was evaluated at 24 h pi while the apoptosis assay was performed at 48 h pi. Levels of pro-inflammatory cytokines: Tnf, Il6, Il10, Il1b and Mcp1 in RAW 264.7 (a) and BMM (b) through RT-PCR. Production of ROS after heat exposure was measured by fluorescence microscopy (c) and spectrometry (d). The infected cells were treated with inhibitors of mitochondrial respiratory chain (TTFA) or NADPH oxidase (DI) (e). The apoptosis was evaluated by activation of caspase-3 (f) and flow cytometry (g) after heat exposure. The intensity of FITC was also evaluated from random 10,000 cells (h). Data represent the mean ± SD of triplicate experiments. Asterisks indicate significant differences (p < 0.01). (Scale bar = 20 μm)