Data is available on the interaction between microorganism-associated molecular patterns and individual toll-like receptors on dendritic cells that turn on signal transduction pathways leading to the activation of different transcription factors. Microbial invasions are perceived by pattern recognition receptor (PRR)-expressing cells of the innate immune system. PRRs bind a number of microbial products collectively referred to as microorganism-associated molecular patterns (MAMPs). Toll-like receptors (TLRs) are the best-characterized PRRs. The interaction of MAM...

We have microarray data on macrophages and DC from different genetic backgrounds of mice, stimulated with CpG, in the presence or absence of a MEK inhibitor, and further data on the role of certain MAP kinases in the regulation of IL-10 and IL-12, IFN-gamma production. Using pharmacological inhibitors, or macrophages and DC from mice carrying a null mutation in MAP kinase signalling molecule(s), we have evidence for a role of certain MAP kinases in the regulation of IL-10 and IL-12, IFN-gamma production. The work on the role of the MAP kinases, TPL-2 and ERK in u...

We investigated and obtained data on the interactions between human monocyte derived DCs (MDDC), generated in the presence of GM-CSF and IL-4 (IL-4 DC), and antigen-stimulated circulating gamma delta T lymphocytes, bearing the Vgamma2 TCR. Co-cultures of IFN-DCs with zoledronate-stimulated gamma delta T lymphocytes were also performed. gamma delta T-cell activation, as demonstrated by their up-modulation of activation marker expression levels (e.g. CD25 and CD69), was observed. These studies demonstrated for the first time the existence of a bidirectional activatin...

We have obtained data on a possible cooperation between myeloid and plasmacytoid DCs in response to different microbial stimuli and a characterization of the mechanisms of this cooperation. For this, we have studied co-culture of mDCs and pDCs in response to CpGs, LPS and bacterial particles. Taking advantage on the fact that human myeloid and plasmacitoid DC show a selective response to LPS and CpG respectively, we have previously identified a cross-talk between two type of cells. Indeed it was observed that LPS-stimulated mDCs are able to induce up-regulation o...

We are currently performing gene silencing experiments in human monocyte-derived DCs (MDDCs) with the dual aim of generating relevant knowledge and data on (i) the transcriptional regulation of DC differentiation, and (ii) the safe and rationale in vitro manipulation of gene expression in DCs. One of the two different approaches that have been set up in parallel is: Lentiviral transduction of shRNAs: to this aim, shRNA specific for IRF4 and STAT3 are in the process to be cloned into the pLVTHM lentiviral vector (obtained from the repository Addgene). This v...

We have data on a novel innate signalling pathway in DC. Using curdlan as a specific agonist of dectin-1, we have shown that this C-type lectin couple to Syk kinase leads to activation of ERK, JNK and p38 MAPKs, as well as NF-kappaB. However, signalling terminates rapidly once the receptor is internalised, thereby explaining previous observations that large, non-phagocytisable Dectin-1 ligands are much more potent agonists than smaller ones. This is the first example of a bona fide signalling pathway from a cell surface innate receptor other than a TLR. We will ...

Data has been obtained on the activation of human DC by Toll like receptor ligands (TLR-L) that modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells. DC derived soluble factors further increase CD1d-restricted iNKT cell activation, as defined by IFN-alpha secretion. Finally, using soluble tetrameric iNKT TCR as a staining reagent we demonstrate specific up-regulation of CD1d-bound ligands upon TLR-mediated APC maturation. The ability of innate stimuli to modulate the lipid profile of DC resulting in iNK...

Our group obtained data on molecular signaling triggered by LPS in dendritic cells and the role of CD14 in the regulation of the DC life cycle through NFAT activation. Toll-like receptors (TLRs) are the best-characterized pattern recognition receptors (PRRs). Individual TLRs recruit diverse combinations of adapter proteins, and thereby trigger signal transduction pathways leading to the activation of various transcription factors, including nuclear factor (NF)-kB, activation protein (AP)-1, and interferon regulatory factors (IRFs). Interleukin-2 (IL-2) is one of th...

We have obtained data on the STAT-1 pathway in response of DC to Th1-inducing stimuli. We have identified some genes differentially expressed in the different conditions. In particular, the most relevant gene upregulated in presence of stimuli typically inducing Th1 responses (LPS, CpG, Poly I:C) but not in presence of the Th2 stimulus, Pam3Cys, was Stat-1. We then evaluated whether STAT-1 was phosphorylated in DC following activation with TLR-dependent Th1 and Th2 stimuli and we found that only in presence of TLR stimuli able to induce Th1 responses STAT-1 was act...

We are currently performing gene silencing experiments in human monocyte-derived DCs (MDDCs) with the dual aim of generating relevant knowledge and data on (i) the transcriptional regulation of DC differentiation, and (ii) the safe and rationale in vitro manipulation of gene expression in DCs. One of the two different approaches that have been set up in parallel is: Transient transfection of synthetic siRNA: to this aim, commercially available siRNA Smart Pool from Dharmacon targeted against the STAT3 transcription factor have been successfully transfected i...

Our group has generated pathway data from human DC treated with pathogen derivatives.

We have been following up on the effort to generate proteomics map data of lysosomes purified from non-activated and activated mouse DCs. The decision to focus on the most important organelle for DC activity has been driven from the need to describe in greater detail the proteins involved in the DC response both qualitatively and quantitatively. The partner has been trying to quantify the relative amount of 400 proteins. They have also indexed better this list and are currently comparing it to a list of proteins present in macrophages lysosomes (in collaboration ...

Pathway-level data have been obtained from a pilot experiment on yeast cells treated with peptides with potential antifungal action. We have compared results from both genomics and proteomics experiments and search for similarities at the level of pathways and have used Eu.Gene to compare the two different data sources demonstrating the feasibility of using this program to this task. Overall correlation between changes in RNA and protein expression was poor but increased when looking at components of immunologically-relevant pathways and Gene ontology terms. The...