Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state

BACKGROUND: Macrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive. RESULTS: RAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6. CONCLUSIONS: These findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge

Collaborative Induction of Inflammatory Responses by Dectin-1 and Toll-like Receptor 2

Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these receptors is likely to cooperate with TLR signaling in defining inflammatory responses. In this report we have examined how dectin-1, a lectin family receptor for β-glucans, collaborates with TLRs in recognizing microbes. Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif–like signaling motif that is tyrosine phosphorylated upon activation. The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G–opsonized particles. Dectin-1 expression enhances TLR-mediated activation of nuclear factor κB by β-glucan–containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor α. Additionally, dectin-1 triggers production of reactive oxygen species, an inflammatory response that is primed by TLR activation. The data demonstrate that collaborative recognition of distinct microbial components by different classes of innate immune receptors is crucial in orchestrating inflammatory responses

Optimization of DNA extraction from human urinary samples for mycobiome community profiling.

IntroductionRecent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi.Materials and methodsWe evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS).ResultsOverall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity.ConclusionsAlterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS

Amphetamine Modulates Excitatory Neurotransmission through Endocytosis of the Glutamate Transporter EAAT3 in Dopamine Neurons

SummaryAmphetamines modify the brain and alter behavior through mechanisms generally attributed to their ability to regulate extracellular dopamine concentrations. However, the actions of amphetamine are also linked to adaptations in glutamatergic signaling. We report here that when amphetamine enters dopamine neurons through the dopamine transporter, it stimulates endocytosis of an excitatory amino acid transporter, EAAT3, in dopamine neurons. Consistent with this decrease in surface EAAT3, amphetamine potentiates excitatory synaptic responses in dopamine neurons. We also show that the process of internalization is dynamin- and Rho-mediated and requires a unique sequence in the cytosolic C terminus of EAAT3. Introduction of a peptide based on this motif into dopamine neurons blocks the effects of amphetamine on EAAT3 internalization and its action on excitatory responses. These data indicate that the internalization of EAAT3 triggered by amphetamine increases glutamatergic signaling and thus contributes to the effects of amphetamine on neurotransmission

The history of the Y chromosome in man

Studies of the Y chromosome over the past few decades have opened a window into the history of our species, through the reconstruction and exploitation of a patrilineal (Y-genealogical) tree based on several hundred single-nucleotide variants (SNVs). A new study validates, refines and extends this tree by incorporating >65,000 Y-linked variants identified in 1,244 men representing worldwide diversity

A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis

Extracellular matrix interactions play essential roles in normal physiology and many pathological processes. While the importance of ECM interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel screening platform capable of measuring phenotypic responses to combinations of ECM molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the ECM-dependent adhesion of tumor-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumor lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8, or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified ECM and integrin interactions that could serve as therapeutic targets

Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse'.

Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects β-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate β-glucan polymers, Dectin-1 signalling is only activated by particulate β-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required