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In vivo TLR9 inhibition attenuates CpG-induced myocardial dysfunction.

Boehm O, Markowski P, van der Giet M, Gielen V, Kokalova A, Brill C, Hoeft A, Baumgarten G, Meyer R, Knuefermann P - Mediators Inflamm. (2013)

Bottom Line: These effects were not observed in TLR9-D mice.Inhibition of TLR9 by the suppressive ODN H154-thioate significantly ameliorated cardiac inflammation, preserved cardiac function, and improved survival.This suppressive ODN was the most efficient inhibitor of the tested substances.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn, Bonn, Germany.

ABSTRACT
The involvement of toll-like receptor 9 (TLR9), a receptor for bacterial DNA, in septic cardiac depression has not been clarified in vivo. Thus, the aim of the study was to test possible TLR9 inhibitors (H154-thioate, IRS954-thioate, and chloroquine) for their ability to protect the cardiovascular system in a murine model of CpG oligodeoxynucleotide- (ODN-) dependent systemic inflammation. Sepsis was induced by i.p. application of the TLR9 agonist 1668-thioate in C57BL/6 wild type (WT) and TLR9-deficient (TLR9-D) mice. Thirty minutes after stimulation TLR9 antagonists were applied i.v. Survival was monitored up to 18 h after stimulation. Cardiac mRNA expression of inflammatory mediators was analyzed 2 h and 6 h after stimulation with 1668-thioate and hemodynamic parameters were monitored at the later time point. Stimulation with 1668-thioate induced a severe sepsis-like state with significant drop of body temperature and significantly increased mortality in WT animals. Additionally, there was a time-dependent increase of inflammatory mediators in the heart accompanied by development of septic heart failure. These effects were not observed in TLR9-D mice. Inhibition of TLR9 by the suppressive ODN H154-thioate significantly ameliorated cardiac inflammation, preserved cardiac function, and improved survival. This suppressive ODN was the most efficient inhibitor of the tested substances.

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(a)–(f) RT-qPCR of mRNA expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the hearts of WT and TLR9-D mice measured 2 and 6 h after application of 1668-thioate. The control ODN 1612-thioate or the TLR9 inhibitors H154- and IRS954-thioate as well as chloroquine were injected 30 min after stimulation. PBS application served as control, and TLR9-D animals (last bar) were stimulated with 1668-thioate + PBS as negative control (mean ± SEM; n = 8/group, *P < 0.05; *also indicates the significant group).
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fig3: (a)–(f) RT-qPCR of mRNA expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the hearts of WT and TLR9-D mice measured 2 and 6 h after application of 1668-thioate. The control ODN 1612-thioate or the TLR9 inhibitors H154- and IRS954-thioate as well as chloroquine were injected 30 min after stimulation. PBS application served as control, and TLR9-D animals (last bar) were stimulated with 1668-thioate + PBS as negative control (mean ± SEM; n = 8/group, *P < 0.05; *also indicates the significant group).

Mentions: Two hours and six hours after 1668-thioate stimulation mediators of inflammation (TNF-α, IL-1β, IL-6) were monitored in cardiac tissue (Figures 3(a)–3(f)). In WT animals, 1668-thioate challenge resulted in a significant upregulation of all three proinflammatory cytokines at both time points with maximal expression at 2 h (Figures 3(a), 3(c), and 3(e)). The additional application of the control oligonucleotide 1612-thioate led to a further increase in IL-1β and IL-6 mRNA expression at 2 h (Figures 3(c) and 3(e)). Four hours later, all three cytokines had decreased by at least 40% (Figures 3(b), 3(d), and 3(f)). The application of H154-thioate diminished the 1668-thioate-dependent cytokine increase of all three mediators 2 h after stimulation. After 6 h this suppression was no longer detectable in neither TNF-α nor Il-1β (Figures 3(b) and 3(d)). Neither IRS954-thioate nor chloroquine suppressed cardiac TNF-α or Il-1β mRNA expression at any time point (Figures 3(a)–3(d)). However, there was a significant downregulation of cardiac IL-6 mRNA expression in the 1668-thioate + IRS954-thioate group 2 h after stimulation (Figure 3(e)). Chloroquine application induced a biphasic response of IL-6, enhancing its expression at 2 h and diminishing it at 6 h (Figures 3(e) and 3(f)). In cardiac tissue of TLR9-D animals, application of 1668-thioate did not influence the mRNA expression of the investigated mediators. In clinical symptoms and survival as well as in cytokine mRNA expression H154-thioate proved to be the most potent inhibitor of TLR9 signaling. Therefore only this TLR9 antagonist was tested with respect to cardiac function.


In vivo TLR9 inhibition attenuates CpG-induced myocardial dysfunction.

Boehm O, Markowski P, van der Giet M, Gielen V, Kokalova A, Brill C, Hoeft A, Baumgarten G, Meyer R, Knuefermann P - Mediators Inflamm. (2013)

(a)–(f) RT-qPCR of mRNA expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the hearts of WT and TLR9-D mice measured 2 and 6 h after application of 1668-thioate. The control ODN 1612-thioate or the TLR9 inhibitors H154- and IRS954-thioate as well as chloroquine were injected 30 min after stimulation. PBS application served as control, and TLR9-D animals (last bar) were stimulated with 1668-thioate + PBS as negative control (mean ± SEM; n = 8/group, *P < 0.05; *also indicates the significant group).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649709&req=5

fig3: (a)–(f) RT-qPCR of mRNA expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in the hearts of WT and TLR9-D mice measured 2 and 6 h after application of 1668-thioate. The control ODN 1612-thioate or the TLR9 inhibitors H154- and IRS954-thioate as well as chloroquine were injected 30 min after stimulation. PBS application served as control, and TLR9-D animals (last bar) were stimulated with 1668-thioate + PBS as negative control (mean ± SEM; n = 8/group, *P < 0.05; *also indicates the significant group).
Mentions: Two hours and six hours after 1668-thioate stimulation mediators of inflammation (TNF-α, IL-1β, IL-6) were monitored in cardiac tissue (Figures 3(a)–3(f)). In WT animals, 1668-thioate challenge resulted in a significant upregulation of all three proinflammatory cytokines at both time points with maximal expression at 2 h (Figures 3(a), 3(c), and 3(e)). The additional application of the control oligonucleotide 1612-thioate led to a further increase in IL-1β and IL-6 mRNA expression at 2 h (Figures 3(c) and 3(e)). Four hours later, all three cytokines had decreased by at least 40% (Figures 3(b), 3(d), and 3(f)). The application of H154-thioate diminished the 1668-thioate-dependent cytokine increase of all three mediators 2 h after stimulation. After 6 h this suppression was no longer detectable in neither TNF-α nor Il-1β (Figures 3(b) and 3(d)). Neither IRS954-thioate nor chloroquine suppressed cardiac TNF-α or Il-1β mRNA expression at any time point (Figures 3(a)–3(d)). However, there was a significant downregulation of cardiac IL-6 mRNA expression in the 1668-thioate + IRS954-thioate group 2 h after stimulation (Figure 3(e)). Chloroquine application induced a biphasic response of IL-6, enhancing its expression at 2 h and diminishing it at 6 h (Figures 3(e) and 3(f)). In cardiac tissue of TLR9-D animals, application of 1668-thioate did not influence the mRNA expression of the investigated mediators. In clinical symptoms and survival as well as in cytokine mRNA expression H154-thioate proved to be the most potent inhibitor of TLR9 signaling. Therefore only this TLR9 antagonist was tested with respect to cardiac function.

Bottom Line: These effects were not observed in TLR9-D mice.Inhibition of TLR9 by the suppressive ODN H154-thioate significantly ameliorated cardiac inflammation, preserved cardiac function, and improved survival.This suppressive ODN was the most efficient inhibitor of the tested substances.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn, Bonn, Germany.

ABSTRACT
The involvement of toll-like receptor 9 (TLR9), a receptor for bacterial DNA, in septic cardiac depression has not been clarified in vivo. Thus, the aim of the study was to test possible TLR9 inhibitors (H154-thioate, IRS954-thioate, and chloroquine) for their ability to protect the cardiovascular system in a murine model of CpG oligodeoxynucleotide- (ODN-) dependent systemic inflammation. Sepsis was induced by i.p. application of the TLR9 agonist 1668-thioate in C57BL/6 wild type (WT) and TLR9-deficient (TLR9-D) mice. Thirty minutes after stimulation TLR9 antagonists were applied i.v. Survival was monitored up to 18 h after stimulation. Cardiac mRNA expression of inflammatory mediators was analyzed 2 h and 6 h after stimulation with 1668-thioate and hemodynamic parameters were monitored at the later time point. Stimulation with 1668-thioate induced a severe sepsis-like state with significant drop of body temperature and significantly increased mortality in WT animals. Additionally, there was a time-dependent increase of inflammatory mediators in the heart accompanied by development of septic heart failure. These effects were not observed in TLR9-D mice. Inhibition of TLR9 by the suppressive ODN H154-thioate significantly ameliorated cardiac inflammation, preserved cardiac function, and improved survival. This suppressive ODN was the most efficient inhibitor of the tested substances.

Show MeSH
Related in: MedlinePlus