SEST - Santé-environnement et Santé-travail

Voies de signalisation des haptènes chimiques dans la cellule dendritique humaine – ALLERGODC

Submission summary

Many chemicals have been shown to be allergens leading to contact or respiratory allergy and therefore pose serious health risks. Dendritic cells (DC) present in peripheral organs such as the skin or the lung are capable to take up and to process allergens. After processing antigens, they differentiate into mature, immunostimulatory cells. In addition of antigen processing, DC need to received signals through Toll-like receptors (TLR) to achieve maturation. These signals are provided by the so-called “danger signals” mainly composed of pathogen-associated molecular patterns recognised by TLRs. Our current work is based on the hypothesis that considering similarities between immunity to simple chemicals and that to infectious agents, it is reasonable to speculate that hapten itself stimulates DC maturation and that chemicals sensitizers could be perceived by DC as “danger signals” with common signaling pathways leading to DC maturation and migration. The main objective of this project is to understand and to characterize how chemical sensitizers activate signaling pathways in human dendritic cells leading to DC activation and maturation. Specific objectives are: • To characterize signaling pathways activated in human DCs using the PepChipKinase technology (kinome analysis) in response to chemical sensitizers (Nickel, formaldehyde, dinitrochlorobenzene), chemical irritants (benzalkonium chloride) and danger signals (LPS from e.coli). • To identify if specific signaling pathways are mobilized in response to chemical sensitizers in comparison to irritant molecules using results from kinome analysis. • To identify what are the signals that are missing when comparing chemical haptens to LPS using results from kinome analysis. • To identify what are the initial molecular targets of chemical sensitizers leading to DC activation. Identification of signaling pathways induced by chemical sensitizers, irritant molecules and danger signals. Our first objective is to characterize signaling pathways activated in human DC using the PepChipKinase technology/kinome analysis (Pepscan systems) in response to chemical sensitizers (Nickel, formaldehyde, dinitrochlorobenzene), chemical irritants (benzalkonium chloride) and danger signals (LPS from e.coli). Molecules will be first tested using the maximal sub-toxic concentration (ie the concentration that induced a maximum of 20% cytotoxicity). Two exposure times will be used: 15 min that will allow the identification of kinases activated immediately after chemical addition and 1 hour that corresponds to optimal MAPK and NFkB activation in our DC model. The cellular model will be human DC obtained from cord blood CD34+ progenitors. CD34+ cells will be differentiated in DC using GM-CSF, TNF-alpha, Flt-3l and SCF (stem cell factor) for 6 days. After 6 days, IL-4 is added for 2 days to obtain more CD1a+ cells. This model was selected due to low individual variations as compared to human DC differentiated from monocytes and to the higher yields obtained. The results obtained using this approach will also allow to: •Characterize signal transduction pathways induced by different types of chemical sensitizers in DC. •To identify if specific signaling pathways are mobilized in response to chemical sensitizers in comparison to irritant molecules. •To identify what are the signals that are missing by comparing results obtained with chemical sensitizers to the one using LPS. The next step will be to address the role of the identified kinases in DC activation induced by chemical sensitizers. Previous work performed by our group and others have identified links between DC phenotype alteration by chemical sensitizers and MAPK and NFkB activation. Consequently, MAPK and NFkB activation will be measured after inhibition of identified kinases to understand links between upstream kinases and downstream pathways and the role of these kinases in DC activation. Kinase inhibition will be performed using pharmacological inhibitors if available and specific enough or with promoter-expressed small hairpin RNA (MISSION™ shRNA). Small hairpin RNA (shRNA) are more appropriate than siRNA due to induction of an interferon (IFN) response by siRNA. Finally, DC phenotype (CD83, CD86, CCR7, CD40, IL-12p40) will be evaluated following inhibition of the main kinases identified. Role of Redox imbalance in DC activation by chemical sensitizers. One of the question, still unresolved, is what are the molecular targets mobilized by chemical sensitizers leading to DC activation. We believed that the PepChipKinase approach will help us in this task. However, in parallel we will also work on the role of redox imbalance in DC activation by chemical sensitizers. Oxygen radicals are recognized as signaling molecules and in this respect they act as mediators of cell apoptosis and as regulators of gene expression by their action on redox-regulated transcription factors, like NFKB and AP-1. We will test in our model the hypothesis that alteration in the GSH/GSSG ratio could mediate signaling by haptens in DC through protein S-thiolation/dethiolation with the hypothesis that the primary target protein of glutathione redox is upstream of MAPK and NF-kB. DC will be treated with nickel, DNCB, formaldehyde or benzalkonium chloride and GSH/GSSG ratio will be measured using the GSSG-reductase 5', 5'-dithio-bis(2-nitrobenzoic acid). The effect of GSHet on nickel, formaldehyde or DNCB-induced DC-maturation marker expression (CCR7, CD86, CD83, HLA-DR, CD40, IL-12p40, IL-6) will be tested. The status of the ASK-1/thioredoxin complex will be evaluated by co-immunoprecipitation since ASK-1 is an upstream activator of P38MAPK and JNK and is activated upon release of oxidized thioredoxin from the complex.

Project coordination

Marc PALLARDY (Université)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Help of the ANR 150,000 euros
Beginning and duration of the scientific project: - 24 Months

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