Cobalt (II) Chloride Regulates the Invasion and Survival of Brucella abortus 544 in RAW 264.7 Cells and B6 Mice.

The effects of Cobalt (II) chloride (CoCl2) in the context of Brucella abortus (B. abortus) infection have not been evaluated so far. Firstly, we found that CoCl2 treatment inhibited the phagocytosis of B. abortus into RAW 264.7 cells. The inhibition of bacterial invasion was regulated by F-actin formation and associated with a reduction in the phosphorylation of ERK1/2 and HIF-1α expression. Secondly, the activation of trafficking regulators LAMP1, LAMP2, and lysosomal enzyme GLA at the transcriptional level activated immune responses, weakening the B. abortus growth at 4 h post-infection (pi). The silencing of HIF-1α increased bacterial survival at 24 h pi. At the same time, CoCl2 treatment showed a significant increase in the transcripts of lysosomal enzyme HEXB and cytokine TNF-α and an attenuation of the bacterial survival. Moreover, the enhancement at the protein level of HIF-1α was induced in the CoCl2 treatment at both 4 and 24 h pi. Finally, our results demonstrated that CoCl2 administration induced the production of serum cytokines IFN-γ and IL-6, which is accompanied by dampened Brucella proliferation in the spleen and liver of treated mice, and reduced the splenomegaly and hepatomegaly. Altogether, CoCl2 treatment contributed to host resistance against B. abortus infection with immunomodulatory effects.

The Key Role of c-Fos for Immune Regulation and Bacterial Dissemination in Brucella Infected Macrophage.

The cellular oncogene c-Fos (c-Fos) is a component of activator protein 1 (AP1), a master transcriptional regulator of cells. The suppression of c-Fos signaling by siRNA treatment resulted in significant induction of TLR4, which subsequently activates p38 and ERK1/2 mitogen-activated protein kinases (MAPKs) and enhances F-actin polymerization, leading to an increase in B. abortus phagocytosis. During B. abortus infection, c-Fos signaling is induced, which activates the downstream innate-immunity signaling cascade for bacterial clearance. The inhibition of c-Fos signaling led to increased production of interleukin 10 (IL-10), which partially suppressed lysosome-mediated killing, resulting in increased survival of B. abortus inside macrophages. We present evidence of the regulatory role played by the c-Fos pathway in proliferation during B. abortus infection; however, this was independent of the anti-Brucella effect of this pathway. Another finding is the essential contribution of c-Fos/TRAIL to infected-cell necrosis, which is a key event in bacterial dissemination. These data provide the mechanism via which c-Fos participates in host defense mechanisms against Brucella infection and in bacterial dissemination by macrophages.

Activation of NF-kB-Mediated TNF-Induced Antimicrobial Immunity Is Required for the Efficient Brucella abortus Clearance in RAW 264.7 Cells.

In this study, we explore the regulatory roles of pro-inflammatory cytokine tumor necrosis factor alpha (TNF) in the innate immunity of macrophages against B. abortus infection. We show that infection of macrophage with B. abortus induces marked expression and secretion of TNF which subsequently binds to TNF receptor 1 (TNFR-1) and activates a downstream signaling cascade of the innate immunity. Blocking of TNF signaling resulted in a notable increase of B. abortus survival which was associated with an increase of anti-inflammatory cytokine interleukin 10 (IL-10), a beneficial effector of Brucella survival, as well as remarkable decrease of reactive oxygen species (ROS) and nitric oxide (NO), antibrucella molecules. However, surprisingly, the interference of TNF did not show any influence on phagolysosome and cell death events. Furthermore, the transcriptional factor NF-kB was found to be a main mediator of TNF signaling when blocking of NF-kB pathway drastically suppressed the TNF-induced brucellacidal effect. Taken together, these findings clearly indicate that the immune cascade activated by TNF/TNFR-1 is required for the sufficient resistance to B. abortus survival in macrophages.