Investigating TLR signaling responses in murine dendritic cells upon bacterial infection.

Innate immune recognition of microbial components is critical for the onset of an appropriate immune response against invading pathogens, in particular by dendritic cells. Toll-like receptors (TLRs) are key in the detection of a variety of microbial stimuli.Here we focus on the methodology used to evaluate the role of TLRs in the process of dendritic cell response to bacterial intracellular infections, using bone marrow-derived dendritic cells (BMDCs) as a model system. This protocol describes how to access the level of activation of BMDCs using standard immunology and biochemistry approaches along with examination of infected cells by immunofluorescence microscopy.

Brucella T4SS: the VIP pass inside host cells.

For many Gram-negative bacteria, like Brucella, the type IV secretion system (T4SS) has a critical role in bacterial virulence. In Brucella, the VirB T4SS permits the injection of bacterial effectors inside host cells, leading to subversion of signaling pathways and favoring bacterial growth and pathogenesis. The virB operon promoter is tightly regulated by a combination of transcriptional activators and repressors that are expressed according to the environmental conditions encountered by Brucella. Recent advances have shed light on the Brucella T4SS regulatory mechanisms and also its substrates. Characterization of the targets and functions of these translocated effectors is underway and will help understand the role of the T4SS in the establishment of a replication niche inside host cells.

Pathogenic brucellae replicate in human trophoblasts.

Brucellae replicate in a vacuole derived from the endoplasmic reticulum (ER) in epithelial cells, macrophages, and dendritic cells. In animals, trophoblasts are also key cellular targets where brucellae efficiently replicate in association with the ER. Therefore, we investigated the ability of Brucella spp. to infect human trophoblasts using both immortalized and primary trophoblasts. Brucella extensively proliferated within different subpopulations of trophoblasts, suggesting that they constitute an important niche in cases where the fetal-maternal barrier is breached. In extravillous trophoblasts (EVTs), B. abortus and B. suis replicated within single-membrane acidic lysosomal membrane-associated protein 1-positive inclusions, whereas B. melitensis replicated in the ER-derived compartment. Furthermore, B. melitensis but not B. abortus nor B. suis interfered with the invasive capacity of EVT-like cells in vitro. Because EVTs are essential for implantation during early stages of pregnancy, the nature of the replication niche may have a central role during Brucella-associated abortion in infected women.

Brucella ß 1,2 cyclic glucan is an activator of human and mouse dendritic cells.

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella ß 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella ß 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella ß 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.