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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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Related in: MedlinePlus

(a)–(c) In vitro evaluation of different doses of TLR9 inhibitors. RAW 264.7 macrophages were stimulated with feces of C57BL/6 WT mice simultaneously with different TLR9 inhibitors for 24 h and TNF-α protein content was monitored via ELISA (mean ± SEM; n = 5; *P < 0.05; *also indicates the significant group).
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fig1: (a)–(c) In vitro evaluation of different doses of TLR9 inhibitors. RAW 264.7 macrophages were stimulated with feces of C57BL/6 WT mice simultaneously with different TLR9 inhibitors for 24 h and TNF-α protein content was monitored via ELISA (mean ± SEM; n = 5; *P < 0.05; *also indicates the significant group).

Mentions: In a pilot study cultured macrophages of the line RAW 264.7 were stimulated with heat-inactivated feces of C57BL/6 mice for 24 h (concentration 106 bacteria/mL). Thereafter, TNF-α protein was measured by ELISA in the supernatant of the cell culture. Stimulation led to a 30-fold increase of TNF-α protein compared with control (Figures 1(a)–1(c)). This increase was used to test the suppressive effect of the TLR9 inhibitors H154-thioate, IRS954-thioate, and chloroquine [8–10]. H154-thioate was applied simultaneously with the polymicrobial stimulus in three different concentrations (50 mg/L, 25 mg/L, and 0.5 mg/L). All applied concentrations of H154 were able to reduce the TNF-α protein significantly in a concentration-dependent manner (Figure 1(a)). Comparable experiments were performed with IRS954-thioate and chloroquine (Figures 1(b) and 1(c)). The effect of IRS954-thioate was less pronounced than that of H154-thioate. The lowest effective concentration for IRS954-thioate was 5 mg/L. Chloroquine was applied in four different concentrations (2.5, 10, 50, and 100 mg/L); the lowest effective concentration was 10 mg/L. In order to ensure efficaciousness of the antagonists a single dose of 8 mg/kg BW of H154- and IRS954-thioate and 10 mg/kg of chloroquine was applied i.v. to the animals.


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)–(c) In vitro evaluation of different doses of TLR9 inhibitors. RAW 264.7 macrophages were stimulated with feces of C57BL/6 WT mice simultaneously with different TLR9 inhibitors for 24 h and TNF-α protein content was monitored via ELISA (mean ± SEM; n = 5; *P < 0.05; *also indicates the significant group).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649709&req=5

fig1: (a)–(c) In vitro evaluation of different doses of TLR9 inhibitors. RAW 264.7 macrophages were stimulated with feces of C57BL/6 WT mice simultaneously with different TLR9 inhibitors for 24 h and TNF-α protein content was monitored via ELISA (mean ± SEM; n = 5; *P < 0.05; *also indicates the significant group).
Mentions: In a pilot study cultured macrophages of the line RAW 264.7 were stimulated with heat-inactivated feces of C57BL/6 mice for 24 h (concentration 106 bacteria/mL). Thereafter, TNF-α protein was measured by ELISA in the supernatant of the cell culture. Stimulation led to a 30-fold increase of TNF-α protein compared with control (Figures 1(a)–1(c)). This increase was used to test the suppressive effect of the TLR9 inhibitors H154-thioate, IRS954-thioate, and chloroquine [8–10]. H154-thioate was applied simultaneously with the polymicrobial stimulus in three different concentrations (50 mg/L, 25 mg/L, and 0.5 mg/L). All applied concentrations of H154 were able to reduce the TNF-α protein significantly in a concentration-dependent manner (Figure 1(a)). Comparable experiments were performed with IRS954-thioate and chloroquine (Figures 1(b) and 1(c)). The effect of IRS954-thioate was less pronounced than that of H154-thioate. The lowest effective concentration for IRS954-thioate was 5 mg/L. Chloroquine was applied in four different concentrations (2.5, 10, 50, and 100 mg/L); the lowest effective concentration was 10 mg/L. In order to ensure efficaciousness of the antagonists a single dose of 8 mg/kg BW of H154- and IRS954-thioate and 10 mg/kg of chloroquine was applied i.v. to the animals.

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