Innate sensing of pathogens elicits protective immune responses through pattern recognition receptors, including Toll-like receptors. Although signaling by Toll-like receptors is regulated at multiple steps, including localization, trafficking, proteolytic cleavage, and phosphorylation, the significance of post-translational modifications and cellular stress response on Toll-like receptor stability and signaling is still largely unknown. In the present study, we investigated the role of cytoplasmic tyrosine motifs in Toll-like receptor-9 stability, proteolytic cleavage, and signaling. We demonstrated that tyrosine phosphorylation is essential for mouse Toll-like receptor-9 protein stability and signaling. Upon inhibition of tyrosine kinases with piceatannol, Toll-like receptor-9 tyrosine phosphorylation induced by CpG deoxyribonucleic acid was inhibited, which correlated with decreased signaling. Furthermore, inhibition of Src kinases with 1-tert-Butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine also inhibited response to CpG deoxyribonucleic acid. Toll-like receptor-9 protein stability was also sensitive to autophagy, the cellular stress response pathway, and infection by a deoxyribonucleic acid virus. Whereas autophagy induced by rapamycin or low serum levels caused a preferential loss of the mature p80 proteolytic cleavage product, infection with herpes simplex virus-1 and induction of cell stress with tunicamycin caused preferential loss of full-length Toll-like receptor-9, which is localized to the endoplasmic reticulum. Our data reveal new information about the stability and signaling of Toll-like receptor-9 and suggest that immune evasion mechanisms may involve targeted loss of innate sensing receptors.
Keywords: CpG DNA; autophagy; inflammation; innate immunity.
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