A novel pathway of rapid TLR-triggered activation of integrin-dependent leukocyte adhesion that requires Rap1 GTPase.
Bottom Line: Consistently, in vivo administration of the TLR2-ligand Pam3CSK4 increased integrin-dependent slow rolling and adhesion to endothelium within minutes, as identified by intravital microscopy in the cremaster model.TLR2 and TLR5 ligation increased β2-integrin affinity, as assessed by the detection of activation-dependent neoepitopes.This novel direct pathway linking initial pathogen recognition by TLRs to rapid β2-integrin activation may critically regulate acute leukocyte infiltration to sites of pathogen invasion.
Affiliation: Department of Clinical Pathobiochemistry, Technische Universität Dresden, 01309 Dresden, Germany Institute of Physiology, Technische Universität Dresden, 01309 Dresden, Germany firstname.lastname@example.org.Show MeSH
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Mentions: Having found that TLR2 and TLR5 ligation induced β2-integrin activation and β2-integrin–dependent leukocyte adhesion in a Rap1-dependent manner, we continued to consider the signaling pathways between TLR ligation and Rap1 activation. ROS production through Nox2 NADPH oxidase activation has been proposed as a downstream event to TLR stimulation (Martinon et al., 2010). To investigate the involvement of ROS production in Rap1 and β2-integrin activation caused by TLR2 and TLR5 ligation, we initially confirmed that treatment with HKLM or flagellin induced ROS production in THP-1 myelomonocytes, as assessed by flow cytometry (Figure 4A). We further studied the effect of Nox2 inhibition in TLR2- and TLR5-induced adhesion of THP-1 cells to ICAM-1. In particular, siRNA-mediated NOX2 gene silencing (Supplemental Figure S3B), but not control siRNA, blocked cell adhesion to ICAM-1 downstream of TLR ligation (Figure 4B) by preventing TLR2-induced Rap1 activation (Figure 4, C and D). In addition, we observed that treatment with diphenyleneiodonium (DPI), a NADPH oxidase inhibitor, significantly blocked TLR2- and TLR5-induced cell adhesion to ICAM-1 (Supplemental Figure S5). These data suggest that Nox2-dependent ROS generation is a signaling intermediate between TLR2 and TLR5 ligation and Rap1-dependent β2-integrin activation.
Affiliation: Department of Clinical Pathobiochemistry, Technische Universität Dresden, 01309 Dresden, Germany Institute of Physiology, Technische Universität Dresden, 01309 Dresden, Germany email@example.com.