Omp25-dependent engagement of SLAMF1 by Brucella abortus in dendritic cells limits acute inflammation and favours bacterial persistence in vivo.

The strategies by which intracellular pathogenic bacteria manipulate innate immunity to establish chronicity are poorly understood. Here, we show that Brucella abortus outer membrane protein Omp25 specifically binds the immune cell receptor SLAMF1 in vitro. The Omp25-dependent engagement of SLAMF1 by B. abortus limits NF-κB translocation in dendritic cells (DCs) with no impact on Brucella intracellular trafficking and replication. This in turn decreases pro-inflammatory cytokine secretion and impairs DC activation. The Omp25-SLAMF1 axis also dampens the immune response without affecting bacterial replication in vivo during the acute phase of Brucella infection in a mouse model. In contrast, at the chronic stage of infection, the Omp25/SLAMF1 engagement is essential for Brucella persistence. Interaction of a specific bacterial protein with an immune cell receptor expressed on the DC surface at the acute stage of infection is thus a powerful mechanism to support microbe settling in its replicative niche and progression to chronicity.

COX-2 Inhibition Reduces Brucella Bacterial Burden in Draining Lymph Nodes.

Brucella is a Gram-negative facultative intracellular bacterium responsible for a chronic disease known as brucellosis, the most widespread re-emerging zoonosis worldwide. Establishment of a Th1-mediated immune response characterized by the production of IL-12 and IFNγ is essential to control the disease. Leukotrienes derived from arachidonic acid (AA) metabolism are known to negatively regulate a protective Th1 immune response against bacterial infections. Here, using genomics approaches we demonstrate that Brucella abortus strongly stimulates the prostaglandin (PG) pathway in dendritic cells (DC). We also show an induction of AA production by infected cells. This correlates with the expression of Ptgs2, a gene encoding the downstream cyclooxygenase-2 (COX-2) enzyme in infected DC. By comparing different infection routes (oral, intradermal, intranasal and conjunctival), we identified the intradermal inoculation route as the more potent in inducing Ptgs2 expression but also in inducing a local inflammatory response in the draining cervical lymph nodes (CLN). NS-398, a specific inhibitor of COX-2 enzymatic activity decreased B. melitensis burden in the CLN after intradermal infection. This effect was accompanied by a decrease of Il10 and a concomitant increase of Ifng expression. Altogether, these results suggest that Brucella has evolved to take advantage of the PG pathway in the harsh environment of the CLN in order to persist and subvert immune responses. This work also proposes that novel strategies to control brucellosis may include the use of COX-2 inhibitors.

Brucella discriminates between mouse dendritic cell subsets upon in vitro infection.

Brucella is a Gram-negative bacterium responsible for brucellosis, a worldwide re-emerging zoonosis. Brucella has been shown to infect and replicate within Granulocyte macrophage colony-stimulating factor (GMCSF) in vitro grown bone marrow-derived dendritic cells (BMDC). In this cell model, Brucella can efficiently control BMDC maturation. However, it has been shown that Brucella infection in vivo induces spleen dendritic cells (DC) migration and maturation. As DCs form a complex network composed by several subpopulations, differences observed may be due to different interactions between Brucella and DC subsets. Here, we compare Brucella interaction with several in vitro BMDC models. The present study shows that Brucella is capable of replicating in all the BMDC models tested with a high infection rate at early time points in GMCSF-IL15 DCs and Flt3l DCs. GMCSF-IL15 DCs and Flt3l DCs are more activated than the other studied DC models and consequently intracellular bacteria are not efficiently targeted to the ER replicative niche. Interestingly, GMCSF-DC and GMCSF-Flt3l DC response to infection is comparable. However, the key difference between these 2 models concerns IL10 secretion by GMCSF DCs observed at 48 h post-infection. IL10 secretion can explain the weak secretion of IL12p70 and TNFα in the GMCSF-DC model and the low level of maturation observed when compared to GMCSF-IL15 DCs and Flt3l DCs. These models provide good tools to understand how Brucella induce DC maturation in vivo and may lead to new therapeutic design using DCs as cellular vaccines capable of enhancing immune response against pathogens.

Subversion of mouse dendritic cell subset function by bacterial pathogens.

Dendritic cells (DCs) play an important role as sentinels of the immune system in initiating and controlling the quality of adaptive immune responses. Located at entry points of the host they can sense and alert the body from dangers such as infection by pathogenic bacteria. Considering their strategic localization it is not surprising that DCs have evolved in a series of DC subtypes, which are well adapted to their microenvironment. Nowadays, the advent of the identification of specific DC subtypes has opened the way for the study of pathogen-DCs interactions and the involved mechanisms of these interactions. Due to key aspect of DCs, several bacterial pathogens have taken advantage of these cells and developed mechanisms to subvert DC function and thereby evade the immune system. This review brings recent insights into DC-pathogenic bacteria cross-talk using the mouse model of infection with an emphasis on DC subtypes.

RUN and FYVE domain-containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4.

Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain-containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17-positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4-treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells.

Reversible lipid accumulation and associated division arrest of Mycobacterium avium in lipoprotein-induced foamy macrophages may resemble key events during latency and reactivation of tuberculosis.

During the dormant phase of tuberculosis, Mycobacterium tuberculosis persists in lung granulomas by residing in foamy macrophages (FM) that contain abundant lipid bodies (LB) in their cytoplasm, allowing bacilli to accumulate lipids as intracytoplasmic lipid inclusions (ILI). An experimental model of FM is presented where bone marrow-derived mouse macrophages are infected with M. avium and exposed to very-low-density lipoprotein (VLDL) as a lipid source. Quantitative analysis of detailed electron microscope observations showed the following results. (i) Macrophages became foamy, and mycobacteria formed ILI, for which host triacylglycerides, rather than cholesterol, was essential. (ii) Lipid transfer occurred via mycobacterium-induced fusion between LB and phagosomes. (iii) Mycobacteria showed a thinned cell wall and became elongated but did not divide. (iv) Upon removal of VLDL, LB and ILI declined within hours, and simultaneous resumption of mycobacterial division restored the number of mycobacteria to the same level as that found in untreated control macrophages. This showed that the presence of ILI resulted in a reversible block of division without causing a change in the mycobacterial replication rate. Fluctuation between ILI either partially or fully extending throughout the mycobacterial cytoplasm was suggestive of bacterial cell cycle events. We propose that VLDL-driven FM constitute a well-defined cellular system in which to study changed metabolic states of intracellular mycobacteria that may relate to persistence and reactivation of tuberculosis.

Cervical Lymph Nodes as a Selective Niche for Brucella during Oral Infections.

Cervical lymph nodes (CLN) are the first lymph nodes encountered by material taking the oral route. To study their role in orally acquired infections, we analyzed 307 patients of up to 14 years treated in the university clinic of Skopje, Macedonia, for brucellosis, a zoonotic bacterial disease frequently acquired by ingestion of contaminated dairy products. From these children, 36% had lymphadenopathy. Among orally infected children, lymphadenopathy with CLN being the only lymph nodes affected was significantly more frequent as compared to those infected by contact with animals (83% vs. 63%), suggesting a possible involvement of CLN during orally acquired human brucellosis. Using a murine model where bacteria are delivered into the oral cavity, we show that Brucella quickly and selectively colonize the CLN where they proliferate and persist over long periods of time for up to 50 days post-infection. A similar efficient though less specific drainage to CLN was found for Brucella, Salmonella typhimurium and fluorescent microspheres delivered by gavage, a pathway likely representing a mixed infection mode of intragastric and oral infection, suggesting a central pathway of drained material. Microspheres as well as bacteria drained to CLN predominately reside in cells expressing CD68 and no or low levels of CD11c. Even though no systemic response could be detected, Brucella induced a locally restricted inflammatory reaction with increased expression levels of interferon γ, interleukin (IL)-6, IL-12, granzyme B and a delayed induction of Nos2. Inflammation led to pronounced lymphadenopathy, infiltration of macrophages/monocytes expressing high levels of major histocompatibility complex II and to formation of epitheloid granulomas. Together, these results highlight the role of CLN in oral infections as both, an initial and efficient trap for bacterial invaders and as possible reservoir for chronic pathogens. They likewise cast a new light on the significance of oral routes for means of vaccination.