Activation of TLR-9 Induces IL-8 Secretion through Peroxynitrite Signaling in Human Neutrophils

Self Cite

TLR9 detects bacterial DNA (CpG DNA) and elicits both innate and adoptive immunity. Recent evidence indicates that TLR9 is expressed in more diverse cell types than initially thought. In this study, we report that HUVECs constitutively express TLR9 and selectively recognize unmethylated CpG motifs in bacterial DNA and synthetic immune stimulatory CpG oligodeoxynucleotides. HUVECs respond to CpG DNA with rapid phosphorylation of IκB-α and NF-κB-mediated gene transcription and surface expression of the adhesion molecules ICAM-1 and E-selectin independent of MAPK signaling. The telomere-derived TLR9 inhibitory oligonucleotide 5′-TTT AGG GTT AGG GTT AGG G-3′, agents that block endosomal acidification such as chloroquine and bafilomycin A, and NF-κB inhibitors abrogated CpG DNA-induced signaling. HUVEC activation by CpG DNA led to markedly enhanced neutrophil adhesion under nonstatic conditions that was further enhanced when neutrophils were stimulated with CpG DNA. The adhesive interactions were blocked by Abs against CD18 and, to a lesser degree, by anti-E-selectin and anti-L-selectin Abs. Our findings demonstrate that bacterial DNA promotes β2 integrin and E-selectin-mediated HUVEC-neutrophil adherence, and indicate the ability of CpG DNA to initiate and/or maintain the inflammatory response.

Section: Discussionmentioning

confidence: 53%

mentioning

confidence: 99%

Bacterial DNA Activates Endothelial Cells and Promotes Neutrophil Adherence through TLR9 Signaling

Kebir

József

Pan

et al. 2009

Self Cite

“…Of note, despite expression of TLR9 by B cells, pDC, and neutrophils (43,44), addition of bacterial DNA (derived from E. coli) did not induce secretion of cytokines in human whole blood (data not shown and Ref. 48). The kinetics of our early transient LPS tolerance model differ from classical LPS tolerance models in experimental animals, which are characterized by an early cellular-mediated phase, which dissipates after 3-5 days, and a late Ab-mediated phase (6,49).…”

Section: Discussionmentioning

confidence: 99%

In Vivo Lipopolysaccharide Exposure of Human Blood Leukocytes Induces Cross-Tolerance to Multiple TLR Ligands

Vos

Pater

Pangaart

et al. 2009

In vitro and in vivo experiments in mice have shown that exposure of cells to the TLR4 ligand LPS induces tolerance toward a second exposure to LPS and induces cross-tolerance to certain other TLR ligands. Recently, we found that LPS tolerance in experimental human endotoxemia and Gram-negative sepsis is associated with elevated levels of IL-1R-associated kinase M, an intracellular negative regulator of MyD88-dependent TLR signaling. In the present study, we investigated whether in vivo exposure of humans to LPS induces tolerance in circulating leukocytes to other TLR agonists that rely either on MyD88- dependent or on MyD88-independent signaling. Analysis of TNF, IL-1β, IL-6, and IL-10 levels in whole blood demonstrated that leukocytes were hyporesponsive to ex vivo LPS restimulation 3–8 h after i.v. LPS injection (4 ng/kg). Reduced cytokine release during the same interval was also observed in whole blood further stimulated with MyD88-dependent ligands for TLR2, TLR5, and TLR7 or with whole bacteria. Strikingly, blood leukocytes were also tolerant to a ligand for TLR3, which signals solely through a MyD88-independent (Toll IL-1R domain-containing adaptor-inducing IFN-β (TRIF)-dependent) pathway. The hyporesponsiveness of leukocytes to TLR3 ligation was associated with reduced rather than increased levels of the recently identified TRIF inhibitor SARM. Taken together, these data indicate that systemic LPS challenge of human volunteers induces cross-tolerance to multiple TLR ligands that signal in a MyD88-dependent or MyD88-independent manner and suggest that LPS exposure of human blood leukocytes may hamper the inflammatory response to various microbial components.

“…Studies performed with human neutrophils by József et al (46,47) showed that bacterial DNA moderately delays apoptosis and induces IL-8 secretion through a CpG-and TLR9-dependent mechanism. Their studies were based on results obtained by using endosomal acidification inhibitors such as chloroquine and bafilomycin A and CpG-methylated DNA but did not include the use of TLR9 knockout mice.…”

Section: Discussionmentioning

confidence: 99%

Neutrophil Signaling Pathways Activated by Bacterial DNA Stimulation

Álvarez

Bass

Geffner

et al. 2006

We have previously shown that bacterial DNA activates human neutrophils in a CpG-independent manner. In this study, we have characterized the signaling pathways involved in the activation mechanism. We found that p38 MAPK, ERK1/2, and JNK pathways, as well as the PI3K/Akt pathway, are activated by bacterial DNA. We also determined that bacterial DNA induces NF-κB and AP-1 activation. When analyzing the role of these pathways on neutrophil functions, we observed that up-regulation of CD11b triggered by bacterial DNA was decreased by pharmacological inhibitors of the p38 MAPK, ERK1/2, and JNK, whereas stimulation of IL-8 release was dependent on p38, ERK1/2, and NF-κB. Moreover, we found that IL-8 production was markedly enhanced by inhibition of JNK, suggesting that this pathway negatively modulates NF-κB-dependent transcription. We also observed that bacterial DNA stimulated IL-1R-associated kinase-1 kinase activity and its partial degradation. Finally, we determined that bacterial DNA stimulated CD11b up-regulation in TLR9−/− but not in MyD88−/− mouse neutrophils, supporting that bacterial DNA induces neutrophil activation through a TLR9-independent and MyD88-dependent pathway.