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A novel pathway of rapid TLR-triggered activation of integrin-dependent leukocyte adhesion that requires Rap1 GTPase.

Chung KJ, Mitroulis I, Wiessner JR, Zheng YY, Siegert G, Sperandio M, Chavakis T - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pathobiochemistry, Technische Universität Dresden, 01309 Dresden, Germany Institute of Physiology, Technische Universität Dresden, 01309 Dresden, Germany kyoung-jin.chung@uniklinikum-dresden.de.

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Nox2-dependent ROS production mediates TLR-dependent cell adhesion by inducing Rap1 activation. (A) ROS levels in THP-1 cells treated with HKLM (TLR2 ligand), flagellin (TLR5 ligand), or ODN2006 (TLR9 ligand) were assessed by flow cytometry using CellROX green reagent. Data are the mean MFI ± SEM (n = 4 independent experiments). *p < 0.05. Student's t test was used for statistical analysis. (B) Adhesion of THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Nox2 to immobilized ICAM-1 in the absence or presence of the indicated TLR ligands. Data expressed as relative adhesion. Data are shown as mean ± SEM (n = 4 independent experiments). One-way ANOVA with Bonferroni posthoc analysis (asterisk and section sign [§] denote significance of the posthoc test; the latter symbol indicates comparison with unstimulated cells transfected with control siRNA). (C) Rap1-GTP (activated Rap1) levels in THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Nox2 (siRNA) and treated with HKLM (TLR2). Data derived from one representative experiment. (D) Densitometric analysis of immunoblots demonstrating that Nox2 knockdown blocks the TLR2-induced Rap1 activation (n = 4 independent experiments). *p < 0.05. Student's t test was used for statistical analysis.
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Figure 4: Nox2-dependent ROS production mediates TLR-dependent cell adhesion by inducing Rap1 activation. (A) ROS levels in THP-1 cells treated with HKLM (TLR2 ligand), flagellin (TLR5 ligand), or ODN2006 (TLR9 ligand) were assessed by flow cytometry using CellROX green reagent. Data are the mean MFI ± SEM (n = 4 independent experiments). *p < 0.05. Student's t test was used for statistical analysis. (B) Adhesion of THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Nox2 to immobilized ICAM-1 in the absence or presence of the indicated TLR ligands. Data expressed as relative adhesion. Data are shown as mean ± SEM (n = 4 independent experiments). One-way ANOVA with Bonferroni posthoc analysis (asterisk and section sign [§] denote significance of the posthoc test; the latter symbol indicates comparison with unstimulated cells transfected with control siRNA). (C) Rap1-GTP (activated Rap1) levels in THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Nox2 (siRNA) and treated with HKLM (TLR2). Data derived from one representative experiment. (D) Densitometric analysis of immunoblots demonstrating that Nox2 knockdown blocks the TLR2-induced Rap1 activation (n = 4 independent experiments). *p < 0.05. Student's t test was used for statistical analysis.

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.


A novel pathway of rapid TLR-triggered activation of integrin-dependent leukocyte adhesion that requires Rap1 GTPase.

Chung KJ, Mitroulis I, Wiessner JR, Zheng YY, Siegert G, Sperandio M, Chavakis T - Mol. Biol. Cell (2014)

Nox2-dependent ROS production mediates TLR-dependent cell adhesion by inducing Rap1 activation. (A) ROS levels in THP-1 cells treated with HKLM (TLR2 ligand), flagellin (TLR5 ligand), or ODN2006 (TLR9 ligand) were assessed by flow cytometry using CellROX green reagent. Data are the mean MFI ± SEM (n = 4 independent experiments). *p < 0.05. Student's t test was used for statistical analysis. (B) Adhesion of THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Nox2 to immobilized ICAM-1 in the absence or presence of the indicated TLR ligands. Data expressed as relative adhesion. Data are shown as mean ± SEM (n = 4 independent experiments). One-way ANOVA with Bonferroni posthoc analysis (asterisk and section sign [§] denote significance of the posthoc test; the latter symbol indicates comparison with unstimulated cells transfected with control siRNA). (C) Rap1-GTP (activated Rap1) levels in THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Nox2 (siRNA) and treated with HKLM (TLR2). Data derived from one representative experiment. (D) Densitometric analysis of immunoblots demonstrating that Nox2 knockdown blocks the TLR2-induced Rap1 activation (n = 4 independent experiments). *p < 0.05. Student's t test was used for statistical analysis.
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Related In: Results  -  Collection

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Figure 4: Nox2-dependent ROS production mediates TLR-dependent cell adhesion by inducing Rap1 activation. (A) ROS levels in THP-1 cells treated with HKLM (TLR2 ligand), flagellin (TLR5 ligand), or ODN2006 (TLR9 ligand) were assessed by flow cytometry using CellROX green reagent. Data are the mean MFI ± SEM (n = 4 independent experiments). *p < 0.05. Student's t test was used for statistical analysis. (B) Adhesion of THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Nox2 to immobilized ICAM-1 in the absence or presence of the indicated TLR ligands. Data expressed as relative adhesion. Data are shown as mean ± SEM (n = 4 independent experiments). One-way ANOVA with Bonferroni posthoc analysis (asterisk and section sign [§] denote significance of the posthoc test; the latter symbol indicates comparison with unstimulated cells transfected with control siRNA). (C) Rap1-GTP (activated Rap1) levels in THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Nox2 (siRNA) and treated with HKLM (TLR2). Data derived from one representative experiment. (D) Densitometric analysis of immunoblots demonstrating that Nox2 knockdown blocks the TLR2-induced Rap1 activation (n = 4 independent experiments). *p < 0.05. Student's t test was used for statistical analysis.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pathobiochemistry, Technische Universität Dresden, 01309 Dresden, Germany Institute of Physiology, Technische Universität Dresden, 01309 Dresden, Germany kyoung-jin.chung@uniklinikum-dresden.de.

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Related in: MedlinePlus