Bacillus Calmette-Guérin-Trained Macrophages Elicit a Protective Inflammatory Response against the Pathogenic Bacteria Brucella abortus.

The bacillus Calmette-Guérin (BCG) can elicit enhanced innate immune responses against a wide range of infections, known as trained immunity. Brucella abortus is the causative agent of brucellosis, a debilitating disease that affects humans and animals. In this study, we demonstrate that C57BL/6 mouse bone marrow-derived macrophages under BCG training enhance inflammatory responses against B. abortus. BCG-trained macrophages showed increased MHC class II and CD40 expression on the cell surface and higher IL-6, IL-12, and IL-1ß production. The increase in IL-1ß secretion was accompanied by enhanced activation of canonical and noncanonical inflammasome platforms. We observed elevated caspase-11 expression and caspase-1 processing in BCG-trained macrophages in response to B. abortus compared with untrained cells. In addition, these BCG-trained cells showed higher NLRP3 expression after B. abortus infection. From a metabolic point of view, signaling through the Akt/mammalian target of rapamycin/S6 kinase pathway was also enhanced. In addition, BCG training resulted in higher inducible NO synthase expression and nitrite production, culminating in an improved macrophage-killing capacity against intracellular B. abortus. In vivo, we monitored a significant reduction in the bacterial burden in organs from BCG-trained C57BL/6 mice when compared with the untrained group. In addition, previous BCG immunization of RAG-1-deficient mice partially protects against Brucella infection, suggesting the important role of the innate immune compartment in this scenario. Furthermore, naive recipient mice that received BM transfer from BCG-trained donors showed greater resistance to B. abortus when compared with their untrained counterparts. These results demonstrate that BCG-induced trained immunity in mice results in better control of intracellular B. abortus in vivo and in vitro.

Magnetotactic bacteria as potential sources of bioproducts.

Magnetotactic bacteria (MTB) produce intracellular organelles called magnetosomes which are magnetic nanoparticles composed of magnetite (Fe3O4) or greigite (Fe3S4) enveloped by a lipid bilayer. The synthesis of a magnetosome is through a genetically controlled process in which the bacterium has control over the composition, direction of crystal growth, and the size and shape of the mineral crystal. As a result of this control, magnetosomes have narrow and uniform size ranges, relatively specific magnetic and crystalline properties, and an enveloping biological membrane. These features are not observed in magnetic particles produced abiotically and thus magnetosomes are of great interest in biotechnology. Most currently described MTB have been isolated from saline or brackish environments and the availability of their genomes has contributed to a better understanding and culturing of these fastidious microorganisms. Moreover, genome sequences have allowed researchers to study genes related to magnetosome production for the synthesis of magnetic particles for use in future commercial and medical applications. Here, we review the current information on the biology of MTB and apply, for the first time, a genome mining strategy on these microorganisms to search for secondary metabolite synthesis genes. More specifically, we discovered that the genome of the cultured MTB Magnetovibrio blakemorei, among other MTB, contains several metabolic pathways for the synthesis of secondary metabolites and other compounds, thereby raising the possibility of the co-production of new bioactive molecules along with magnetosomes by this species.

Guanylate-binding protein-5 is involved in inflammasome activation by bacterial DNA but only the cooperation of multiple GBPs accounts for control of Brucella abortus infection.

Introduction: Guanylate-binding proteins (GBPs) are produced in response to pro-inflammatory signals, mainly interferons. The most studied cluster of GBPs in mice is on chromosome 3. It comprises the genes for GBP1-to-3, GBP5 and GBP7. In humans, all GBPs are present in a single cluster on chromosome 1. Brucella abortus is a Gram-negative bacterium known to cause brucellosis, a debilitating disease that affects both humans and animals. Our group demonstrated previously that GBPs present on murine chromosome 3 (GBPchr3) is important to disrupt Brucella-containing vacuole and GBP5 itself is important to Brucella intracellular LPS recognition. In this work, we investigated further the role of GBPs during B. abortus infection. Methods and results: We observed that all GBPs from murine chromosome 3 are significantly upregulated in response to B. abortus infection in mouse bone marrow-derived macrophages. Of note, GBP5 presents the highest expression level in all time points evaluated. However, only GBPchr3-/- cells presented increased bacterial burden compared to wild-type macrophages. Brucella DNA is an important Pathogen-Associated Molecular Pattern that could be available for inflammasome activation after BCV disruption mediated by GBPs. In this regard, we observed reduced IL-1ß production in the absence of GBP2 or GBP5, as well as in GBPchr3-/- murine macrophages. Similar result was showed by THP-1 macrophages with downregulation of GBP2 and GBP5 mediated by siRNA. Furthermore, significant reduction on caspase-1 p20 levels, LDH release and Gasdermin-D conversion into its mature form (p30 N-terminal subunit) was observed only in GBPchr3-/- macrophages. In an in vivo perspective, we found that GBPchr3-/- mice had increased B. abortus burden and higher number of granulomas per area of liver tissue, indicating increased disease severity. Discussion/conclusion: Altogether, these results demonstrate that although GBP5 presents a high expression pattern and is involved in inflammasome activation by bacterial DNA in macrophages, the cooperation of multiple GBPs from murine chromosome 3 is necessary for full control of Brucella abortus infection.

Current Understanding of Bacillus Calmette-Guérin-Mediated Trained Immunity and Its Perspectives for Controlling Intracellular Infections.

The bacillus Calmette-Guérin (BCG) is an attenuated bacterium derived from virulent Mycobacterium bovis. It is the only licensed vaccine used for preventing severe forms of tuberculosis in children. Besides its specific effects against tuberculosis, BCG administration is also associated with beneficial non-specific effects (NSEs) following heterologous stimuli in humans and mice. The NSEs from BCG could be related to both adaptive and innate immune responses. The latter is also known as trained immunity (TI), a recently described biological feature of innate cells that enables functional improvement based on metabolic and epigenetic reprogramming. Currently, the mechanisms related to BCG-mediated TI are the focus of intense research, but many gaps are still in need of elucidation. This review discusses the present understanding of TI induced by BCG, exploring signaling pathways that are crucial to a trained phenotype in hematopoietic stem cells and monocytes/macrophages lineage. It focuses on BCG-mediated TI mechanisms, including the metabolic-epigenetic axis and the inflammasome pathway in these cells against intracellular pathogens. Moreover, this study explores the TI in different immune cell types, its ability to protect against various intracellular infections, and the integration of trained innate memory with adaptive memory to shape next-generation vaccines.

MyD88-dependent BCG immunotherapy reduces tumor and regulates tumor microenvironment in bladder cancer murine model.

Bacillus Calmette-Guerin (BCG) is the only FDA approved first line therapy for patients with nonmuscle invasive bladder cancer. The purpose of this study is to better understand the role of innate immune pathways involved in BCG immunotherapy against murine bladder tumor. We first characterized the immunological profile induced by the MB49 mouse urothelial carcinoma cell line. MB49 cells were not able to activate an inflammatory response (TNF-α, IL-6, CXCL-10 or IFN-ß) after the stimulus with different agonists or BCG infection, unlike macrophages. Although MB49 cells are not able to induce an efficient immune response, BCG treatment could activate other cells in the tumor microenvironment (TME). We evaluated BCG intratumoral treatment in animals deficient for different innate immune molecules (STING-/-, cGAS-/-, TLR2-/-, TLR3-/-, TLR4-/-, TLR7-/-, TLR9-/-, TLR3/7/9-/-, MyD88-/-, IL-1R-/-, Caspase1/11-/-, Gasdermin-D-/- and IFNAR-/-) using the MB49 subcutaneous mouse model. Only MyD88-/- partially responded to BCG treatment compared to wild type (WT) mice, suggesting a role played by this adaptor molecule. Additionally, BCG intratumoral treatment regulates cellular infiltrate in TME with an increase of inflammatory macrophages, neutrophils and CD8+ T lymphocytes, suggesting an immune response activation that favors tumor remission in WT mice but not in MyD88-/-. The experiments using MB49 cells infected with BCG and co-cultured with macrophages also demonstrated that MyD88 is essential for an efficient immune response. Our data suggests that BCG immunotherapy depends partially on the MyD88-related innate immune pathway.

Guanylate binding proteins contained in the murine chromosome 3 are important to control mycobacterial infection.

Guanylate binding proteins (GBPs) are important effector molecules of autonomous response induced by proinflammatory stimuli, mainly IFNs. The murine GBPs clustered in chromosome 3 (GBPchr3) contains the majority of human homologous GBPs. Despite intense efforts, mycobacterial-promoted diseases are still a major public health problem. However, the combined importance of GBPchr3 during mycobacterial infection has been overlooked. This study addresses the influence of the GBPchr3 in host immunity against mycobacterial infection to elucidate the relationship between cell-intrinsic immunity and triggering of an efficient anti-mycobacterial immune response. Here we show that all GBPchr3 are up-regulated in lungs of mice during Mycobacterium bovis BCG infection, resembling tissue expression of IFN-γ. Mice deficient in GBPchr3 (GBPchr3-/- ) were more susceptible to infection, displaying diminished expression of autophagy-related genes (LC3B, ULK1, and ATG5) in lungs. Additionally, there was reduced proinflammatory cytokine production complementary to diminished numbers of myeloid cells in spleens of GBPchr3-/- . Higher bacterial burden in GBPchr3-/- animals correlated with increased number of tissue granulomas. Furthermore, absence of GBPchr3 hampered activation and production of TNF-α and IL-12 by dendritic cells. Concerning macrophages, lack of GBPs impaired their antimicrobial function, diminishing autophagy induction and intracellular killing efficiency. In contrast, single GBP2 deficiency did not contribute to in vivo bacterial control. In conclusion, this study shows that GBPchr3 are important not only to stimulate cell-intrinsic immunity but also for inducing an efficient immune response to control mycobacterial infection in vivo.