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Identification de facteurs de régulation de la voie de signalisation TLR3 par crible génétique

Abstract : The fight of the organism against pathogens involves the detection of molecular patterns that are conserved among pathogens. Among the arsenal of receptors capable of recognizing these patterns, there is the family of endosomal Toll-like receptors (TLRs) that are specific for nucleic acids. Among them, TLR3 senses the abnormal presence of double-stranded RNA in the endosomes and initiates a potent innate immune response. Nevertheless, mechanisms governing TLR3 regulation still remain poorly understood. To identify new molecular players involved in the TLR3 pathway, we performed a genome-wide screen using the CRISPR/Cas9 technology using reporter cells expressing GFP when stimulated via TLR3. Mutagenesis was achieved by transducing these cells with the lentiviral GeCKO v2 sgRNA library. Cells were then subjected to sequential rounds of stimulation with poly(I:C) and sorting of the GFP- cells. Enrichments in sgRNA estimated by deep-sequencing identified genes required for TLR3-induced activation of NF-κB. Five genes, including TLR3 itself and the chaperone UNC93B1, known to be critically involved in the TLR3 pathway, were identified by the screen, thus validating our strategy. We further studied the best 40 hits. Among the hits confirmed, we focused on AhR (aryl hydrocarbon receptor). Depletion of AhR had a dual effect on the TLR3 response, abrogating the IL-8 production and enhancing the IP-10 release. Interestingly, in primary human macrophages exposed to poly(I:C), AhR activation enhanced IL-8 and inhibited the IP-10 one. Overall, AhR appears able to modulate the TLR3 response.
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Laurent Zablocki. Identification de facteurs de régulation de la voie de signalisation TLR3 par crible génétique. Immunité innée. Sorbonne Université, 2018. Français. ⟨NNT : 2018SORUS533⟩. ⟨tel-02900255⟩

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