Bifidobacterium breve Exopolysaccharide Blocks Dendritic Cell Maturation and Activation of CD4+ T Cells.

Exopolysaccharide (EPS) is a bacterial extracellular carbohydrate moiety which has been associated with immunomodulatory activity and host protective effects of several gut commensal bacteria. Bifidobacterium breve are early colonizers of the human gastrointestinal tract (GIT) but the role of EPS in mediating their effects on the host has not been investigated for many strains. Here, we characterized EPS production by a panel of human B. breve isolates and investigated the effect of EPS status on host immune responses using human and murine cell culture-based assay systems. We report that B. breve EPS production is heterogenous across strains and that immune responses in human THP-1 monocytes are strain-specific, but not EPS status-specific. Using wild type and isogenic EPS deficient mutants of B. breve strains UCC2003 and JCM7017 we show that EPS had strain-specific divergent effects on cytokine responses from murine bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs). The B. breve UCC2003 EPS negative (EPS-) strain increased expression of cytokine genes (Tnfa, Il6, Il12a, and Il23a) relative to untreated BMDCs and BMDCs treated with wild type strain. B. breve UCC2003 and JCM7017 EPS- strains increased expression of dendritic cell (DC) activation and maturation marker genes (Cd80, Cd83, and Cd86) relative to untreated BMDCs. Consistent with this, BMDCs co-cultured with B. breve UCC2003 and JCM7017 EPS- strains engineered to express OVA antigen activated OVA-specific OT-II CD4+ T-cells in a co-culture antigen-presentation assay while EPS proficient strains did not. Collectively, these data indicate that B. breve EPS proficient strains use EPS to prevent maturation of DCs and activation of antigen specific CD4+ T cells responses to B. breve. This study identifies a new immunomodulatory role for B. breve EPS and suggests it may be important for immune evasion of adaptive immunity by B. breve and contribute to host-microbe mutualism.

Bone Marrow-Derived Macrophage Immortalization of LXR Nuclear Receptor-Deficient Cells.

Macrophages are professional phagocytic cells that play key roles in innate and adaptive immunity, metabolism, and tissue homeostasis. Lipid metabolism is tightly controlled at the transcriptional level, and one of the key players of this regulation in macrophages and other cell types is the LXR subfamily of nuclear receptors (LXRα and LXRß). The use of LXR double knockout (LXR-DKO) macrophages in vitro has yielded extensive benefits in metabolism research, but this technique is hindered by primary macrophage cell expansion capability, which diminishes along terminal cell differentiation process. Here we detail a method to immortalize LXR double knockout bone marrow-derived macrophage cells at an early stage of differentiation, using a retroviral delivery of a combination of murine v-myc and v-raf oncogenes. This methodology enables the generation of autonomous self-renewing macrophages bearing an LXR-DKO genetic background, as a valuable tool for research in lipid metabolism and other LXR receptor-mediated effects.

Analysis of LXR Nuclear Receptor Cistrome Through ChIP-Seq Data Bioinformatics.

Liver X receptors are members of the nuclear receptor superfamily of transcription factors. The LXR genes (NR1H2 and NR1H3) encode for two different proteins referred to as LXRα and LXRß. Each LXR presents diverse tissue distribution but similar target DNA-binding elements and ligands. Both LXRs act as relevant transcriptional regulators of cholesterol metabolism in many tissues. Additionally, LXRs participate in innate immunity and inflammation. Therefore, in order to understand the molecular requirements that operate in LXR-dependent transcription, it is important to decipher LXR genomic binding properties. We have recently performed genome-wide binding analysis of LXR proteins. In this method paper, we describe a detailed computational protocol primarily based on HOMER software package for the analysis of ChIP-seq data.

Common and Differential Transcriptional Actions of Nuclear Receptors Liver X Receptors α and ß in Macrophages.

The liver X receptors α and ß (LXRα and LXRß) are oxysterol-activated transcription factors that coordinately regulate gene expression that is important for cholesterol and fatty acid metabolism. In addition to their roles in lipid metabolism, LXRs participate in the transcriptional regulation of macrophage activation and are considered potent regulators of inflammation. LXRs are highly similar, and despite notable exceptions, most of their reported functions are substantially overlapping. However, their individual genomic distribution and transcriptional capacities have not been characterized. Here, we report a macrophage cellular model expressing equivalent levels of tagged LXRs. Analysis of data from chromatin immunoprecipitation coupled with deep sequencing revealed that LXRα and LXRß occupy both overlapping and exclusive genomic regulatory sites of target genes and also control the transcription of a receptor-exclusive set of genes. Analysis of genomic H3K27 acetylation and mRNA transcriptional changes in response to synthetic agonist or antagonist treatments revealed a putative mode of pharmacologically independent regulation of transcription. Integration of microarray and sequencing data enabled the description of three possible mechanisms of LXR transcriptional activation. Together, these results contribute to our understanding of the common and differential genomic actions of LXRs and their impact on biological processes in macrophages.