Microbial Recognition Via Toll-Like Receptor-Dependent and -Independent Pathways Determines the Cytokine Response of Murine Dendritic Cell Subsets to CD40 Triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8alpha(+) and CD8alpha(-)CD4(-) DC to make IL-12 p70. In contrast, exposure of CD8alpha(+), CD4(+) and CD8alpha(-)CD4(-) DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.

Activation of G-protein-coupled receptor 30 is sufficient to enhance spatial recognition memory in ovariectomized rats

In ovariectomized rats, administration of estradiol, or selective estrogen receptor agonists that activate either the alpha or beta isoforms, have been shown to enhance spatial cognition on a variety of learning and memory tasks, including those that capitalize on the preference of rats to seek out novelty. Although the effects of the putative estrogen G-protein-coupled receptor 30 (GPR30) on hippocampus-based tasks have been reported using food-motivated tasks, the effects of activation of GPR30 receptors on tasks that depend on the preference of rats to seek out spatial novelty remain to be determined. Therefore, the aim of the current study was to determine if short-term treatment of ovariectomized rats with G-1, an agonist for GPR30, would mimic the effects on spatial recognition memory observed following short-term estradiol treatment. In Experiment 1, ovariectomized rats treated with a low dose (1mug) of estradiol 48h and 24h prior to the information trial of a Y-maze task exhibited a preference for the arm associated with the novel environment on the retention trial conducted 48h later. In Experiment 2, treatment of ovariectomized rats with G-1 (25mug) 48h and 24h prior to the information trial of a Y-maze task resulted in a greater preference for the arm associated with the novel environment on the retention trial. Collectively, the results indicated that short-term treatment of ovariectomized rats with a GPR30 agonist was sufficient to enhance spatial recognition memory, an effect that also occurred following short-term treatment with a low dose of estradiol.

Syk-Dependent Cytokine Induction by Dectin-1 Reveals a Novel Pattern Recognition Pathway for C Type Lectins

Pattern-recognition receptors (PRRs) detect molecular signatures of microbes and initiate immune responses to infection. Prototypical PRRs such as Toll-like receptors (TLRs) signal via a conserved pathway to induce innate response genes. In contrast, the signaling pathways engaged by other classes of putative PRRs remain ill defined. Here, we demonstrate that the β-glucan receptor Dectin-1, a yeast binding C type lectin known to synergize with TLR2 to induce TNFα and IL-12, can also promote synthesis of IL-2 and IL-10 through phosphorylation of the membrane proximal tyrosine in the cytoplasmic domain and recruitment of Syk kinase. syk−/− dendritic cells (DCs) do not make IL-10 or IL-2 upon yeast stimulation but produce IL-12, indicating that the Dectin-1/Syk and Dectin-1/TLR2 pathways can operate independently. These results identify a novel signaling pathway involved in pattern recognition by C type lectins and suggest a potential role for Syk kinase in regulation of innate immunity.

Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8α+ DC correlates with unresponsiveness to imidazoquinolines

Toll-like receptors (TLR) recognize microbial and viral patterns and activate dendritic cells (DC). TLR distribution among human DC subsets is heterogeneous: plasmacytoid DC (PDC) express TLR1, 7 and 9, while other DC types do not express TLR9 but express other TLR. Here, we report that mRNA for most TLR is expressed at similar levels by murine splenic DC sub-types, including PDC, but that TLR3 is preferentially expressed by CD8α+ DC while TLR5 and TLR7 are selectively absent from the same subset. Consistent with the latter, TLR7 ligand activates CD8α– DC and PDC, but not CD8α+ DC as measured by survival ex vivo, up-regulation of surface markers and production of IL-12p40. These data suggest that the dichotomy in TLR expression between plasmacytoid and non-plasmacytoid DC is not conserved between species. However, lack of TLR7 expression could restrict the involvement of CD8α+ DC in recognition of certain mouse pathogens.

Alterations in receptor binding properties of recent human influenza H3N2 viruses are associated with reduced natural killer cell lysis of infected cells

Natural killer (NK) cell recognition of influenza virus-infected cells involves hemagglutinin (HA) binding to sialic acid (SA) on activating NK receptors. SA also acts as a receptor for the binding of influenza virus to its target host cells. The SA binding properties of H3N2 influenza viruses have been observed to change during circulation in humans: recent isolates are unable to agglutinate chicken red blood cells and show reduced affinity for synthetic glycopolymers representing SA-alpha-2,3-lactose (3'SL-PAA) and SA-alpha-2,6-N-acetyl lactosamine (6'SLN-PAA) carbohydrates. Here, NK lysis of cells infected with human H3N2 influenza viruses isolated between 1969 and 2003 was analyzed. Cells infected with recent isolates (1999 to 2003) were found to be lysed less effectively than cells infected with older isolates (1969 to 1996). This change occurred concurrently with the acquisition of two new potential glycosylation site motifs in RA. Deletion of the potential glycosylation site motif at 133 to 135 in HA1 from a recent isolate partially restored the agglutination phenotype to a recombinant virus, indicating that the HA-SA interaction is inhibited by the glycosylation modification. Deletion of either of the recently acquired potential glycosylation sites from HA led to increased NK lysis of cells infected with recombinant viruses carrying modified HA. These results indicate that alterations in RA glycosylation may affect NK cell recognition of influenza virus-infected cells in addition to virus binding to host cells.

Supramolecular aggregates between carboxylate anions and an octaaza macrocyclic receptor

The 28-membered octaazamacrocycle Me-2[28]py(2)N(6) was used as a receptor for the molecular recognition of aromatic and aliphatic carboxylate substrates. The receptor-substrate binding behaviour of (H6Me2[28]py(2)N(6))(6+) with an aliphatic (-O2C(CH2)(n)CO2-, n=0 to 4) and an aromatic (phthalate, isophthalate, terephthalate, 4,4'-dibenzoate, benzoate, 3- and 4-nitrobenzoate) series of carboxylate anions was evaluated by H-1 NMR spectroscopy (carried out in DMSO-d(6) at 300 K). Two association constants were found for most of the studied cases, except for 3- and 4-nitrobenzoate for which only K-1 was determined. For oxalate, malonate, benzoate and dibenzoate anions only the beta(2) constants could be obtained. The values of the first association constant cover a range from 2.86 to 3.69 (log units), and the second stepwise constant from 2.15 to 2.89 (also in log units). No special selectivity was found but the highest values were determined for adipate and the lowest for the monoprotic 3- and 4-nitrobenzoates. Single crystal X-ray structures of H6Me2[28]py(2)N(6)(6+) with terephthalate, 1, and 4,4'-dibenzoate (2) were determined showing supramolecular entities with general formula (H6Me2[28]py(2)N(6)).(substrate)(2)(PF6)(2).4H(2)O. These anions are the building blocks of an extensive 3-D network of hydrogen bonds.

Carboxylate anions binding and sensing by a novel tetraazamacrocycle containing ferrocene as receptor

A tetraazamacrocycle containing ferrocene moieties has been synthesized and characterized. The tetraprotonated form of this compound was evaluated as a receptor (R) for anion recognition of several substrates (S), Cl-, PF6-, HSO4-, H2PO4- and carboxylates, such as p-nitrobenzoate (p-nbz(-)), phthalate (ph(2-)), isophthalate (iph(2-)) and dipicolinate (dipic(2-)). H-1 NMR titrations in CD3OD indicated that this receptor is not suitable for recognizing HSO4- and H2PO4-, but weakly binds p-nbz(-), and strongly interacts with ph(2-), dipic(2-), and iph(2-) anions forming 1 : 2 assembled species. The largest beta(2) binding constant was determined for ph(2-), followed by dipic(2-) and finally iph(2-). The effect of the anionic substrates on the electron-transfer process of the ferrocene units of R was evaluated using cyclic voltammetry (CV) and square wave voltammetry (SWV) in methanol solution and 0.1 mol dm(-3) (CH3)(4)NCl as the supporting electrolyte. Titrations of the receptor were undertaken by addition of anion solutions in their tetrabutylammonium or tetramethylammonium forms. The protonated ligand exhibits a reversible voltammogram, which shifts cathodically in the presence of the substrates. The data revealed kinetic constraints in the formation of the receptor/substrate entity for dipic(2-), ph(2-) and iph(2-) anions, but not for p-nbz(-). In spite of the slow kinetics of assembled species formation with the ph(2-) substrate, this anion provides the largest redox-response when the supramolecular entity is formed, followed by dipic(2-), iph(2-) and finally p-nbz(-) anions. This trend is in agreement with the H-1 NMR results and the values of the binding constants. Single crystal X-ray structures of the receptor with PF6-, ph(2-), iph(2-) and p-nbz(-) were carried out and showed that supermolecules with a RS2 stoichiometry are formed with the first three anions, but RS4 with p-nbz(-). In all cases the binding occurs outside the macrocyclic cavity via N-H center dot center dot center dot O=C hydrogen bonds for carboxylate anions and N - H center dot center dot center dot F hydrogen bonds for the PF6- anion, which is in agreement with the solution results. The macrocyclic framework adopts different conformations in order to interact with each substrate having Fe center dot center dot center dot Fe intramolecular distances ranging from 10.125(14) to 12.783(15) angstrom.

Ditopic redox-active polyferrocenyl zinc(II) dithiocarbamate macrocyclic receptors: synthesis, coordination and electrochemical recognition properties

The synthesis of a range of ditopic polyferrocenyl zinc(II) dithiocarbamate macrocyclic receptors containing ferrocene groups on the macrocycle's periphery and/or as part of the cyclic cavity is reported. The assemblies have been characterised by a range of spectroscopic techniques, electrochemical studies and in two cases by X-ray structure determination. The ability of these host systems to bind and sense electrochemically anionic guest species, isonicotinate and benzoate, and neutral 4-picoline guest was examined by H-1 NMR and cyclic voltammetric titration studies. The strongest association was found between the isonicotinate anion and a dinuclear zinc(II) receptor whose macrocyclic cavity is of complementary size to complex this bidentate guest species in a cooperative manner. Cyclic voltammetric studies demonstrated that all receptors can electrochemically sense the binding of isonicotinate and benzoate via significant cathodic perturbations of the respective ferrocene redox couple.

Alterations in receptor binding properties of recent human influenza H3N2 viruses are associated with reduced natural killer cell lysis of infected cells

Natural killer (NK) cell recognition of influenza virus-infected cells involves hemagglutinin (HA) binding to sialic acid (SA) on activating NK receptors. SA also acts as a receptor for the binding of influenza virus to its target host cells. The SA binding properties of H3N2 influenza viruses have been observed to change during circulation in humans: recent isolates are unable to agglutinate chicken red blood cells and show reduced affinity for synthetic glycopolymers representing SA-alpha-2,3-lactose (3'SL-PAA) and SA-alpha-2,6-N-acetyl lactosamine (6'SLN-PAA) carbohydrates. Here, NK lysis of cells infected with human H3N2 influenza viruses isolated between 1969 and 2003 was analyzed. Cells infected with recent isolates (1999 to 2003) were found to be lysed less effectively than cells infected with older isolates (1969 to 1996). This change occurred concurrently with the acquisition of two new potential glycosylation site motifs in RA. Deletion of the potential glycosylation site motif at 133 to 135 in HA1 from a recent isolate partially restored the agglutination phenotype to a recombinant virus, indicating that the HA-SA interaction is inhibited by the glycosylation modification. Deletion of either of the recently acquired potential glycosylation sites from HA led to increased NK lysis of cells infected with recombinant viruses carrying modified HA. These results indicate that alterations in RA glycosylation may affect NK cell recognition of influenza virus-infected cells in addition to virus binding to host cells.

Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC.

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the alpha-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.

The leptin receptor Gln223Arg polymorphism (rs1137101) mediates the postprandial lipaemic response, but only in males.

Objective: An exaggerated postprandial triacylglycerol (TAG) response is an important determinant of cardiovascular disease risk. With increased recognition of the role of leptin in systemic macronutrient metabolism, we used a candidate gene approach to examine the impact of the common leptin receptor (LEPR) Gln223Arg polymorphism (rs1137101) on postprandial lipaemia. Methods and results: Healthy adults (n ¼ 251) underwent a sequential meal postprandial investigation, in which blood samples were taken at regular intervals after a test breakfast (t ¼ 0) and lunch (t ¼ 330 min). Fasting total- and low-density lipoprotein cholesterol were 9% lower in the ArgArg than GlnArg group (P < 0.04), whereas fasting TAG was 27% lower in the ArgArg than GlnGln group (P < 0.02). The magnitude of the postprandial TAG response was also significantly lower in the ArgArg compared with the GlnArg and GlnGln genotypes, with a 26% lower area under the curve (AUC) and incremental AUC in the ArgArg individuals (P � 0.023). Genotype*gender interactions were evident for fasting and postprandial TAG responses (P < 0.05), with the genotype effect only evident in males. Regression analysis indicated that the LEPR genotype and genotype*gender interactions were independent predictors of the TAG AUC, accounting for 6.3% of the variance. Our main findings were replicated in the independent LIPGENE-Cordoba postprandial cohort of metabolic syndrome subjects (n ¼ 75), with a 52% lower TAG AUC in the ArgArg than GlnGln male subjects (P ¼ 0.018). Conclusion: We report for the first time that the common LEPR Gln223Arg genotype is an important predictor of postprandial TAG in males. The mechanistic basis of these associations remains to be determined.

Controversial role of toll-like receptor 4 in adult stem cells

Adult or somatic stem cells are tissue-resident cells with the ability to proliferate, exhibit self-maintenance as well as to generate new cells with the principal phenotypes of the tissue in response to injury or disease. Due to their easy accessibility and their potential use in regenerative medicine, adult stem cells raise the hope for future personalisable therapies. After infection or during injury, they are exposed to broad range of pathogen or damage-associated molecules leading to changes in their proliferation, migration and differentiation. The sensing of such damage and infection signals is mostly achieved by Toll-Like Receptors (TLRs) with Toll-like receptor 4 being responsible for recognition of bacterial lipopolysaccharides (LPS) and endogenous danger-associated molecular patterns (DAMPs). In this review, we examine the current state of knowledge on the TLR4-mediated signalling in different adult stem cell populations. Specifically, we elaborate on the role of TLR4 and its ligands on proliferation, differentiation and migration of mesenchymal stem cells, hematopoietic stem cells as well as neural stem cells. Finally, we discuss conceptual and technical pitfalls in investigation of TLR4 signalling in stem cells.