Heidi Ehrentraut

Bacterial DNA induces myocardial inflammation and reduces cardiomyocyte contractility: role of Toll-like receptor 9

AIMS Myocardial function is severely compromised during sepsis. Several underlying mechanisms have been proposed. The innate immune system, i.e. toll-like receptor (TLR) 2 and 4, significantly contributes to cardiac dysfunction. Little is known regarding TLR9 and its pathogenic ligand bacterial DNA in the myocardium. We therefore studied the role of TLR9 in myocardial inflammation and cardiac contractility. METHODS AND RESULTS Wild-type (WT, C57BL/6) and TLR9-deficient (TLR9-D) mice and isolated cardiomyocytes were challenged with synthetic bacterial DNA (CpG-ODN). Myocardial contractility as well as markers of inflammation/signalling were determined. Isolated cardiomyocytes incorporated fluorescence-marked CpG-ODN. In WT mice, CpG-ODN caused a robust response in hearts demonstrated by increased levels of tumour necrosis factor (TNF-alpha), interleukin (IL)-1beta, IL-6, inducible nitric oxide synthase (iNOS), and nuclear factor kappaB activity. This inflammatory response was absent in TLR9-D mice. Under similar conditions, contractility measurements of isolated ventricular cardiomyocytes demonstrated a TLR9-dependent loss of sarcomeric shortening after CpG-ODN exposure. This observation was iNOS dependent as the application of a specific iNOS inhibitor reversed sarcomeric shortening to normal levels. CONCLUSION Our data suggest that bacterial DNA contributes to myocardial cytokine production and loss of cardiomyocyte contractility via TLR9.

CpG oligonucleotide activates Toll-like receptor 9 and causes lung inflammation in vivo

BackgroundBacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo.MethodsWild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured.ResultsIn WT mice, CpG-ODN induced a strong activation of pulmonary NFκB as well as a significant increase in pulmonary TNF-α and IL-1β mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice.ConclusionThis study suggests that bacterial CpG-ODN causes lung inflammation via TLR9.

CD73+ regulatory T cells contribute to adenosine-mediated resolution of acute lung injury

Acute lung injury (ALI) is characterized by alveolar injury and uncontrolled inflammation. Since most cases of ALI resolve spontaneously, understanding the endogenous mechanisms that promote ALI resolution is important to developing effective therapies. Previous studies have implicated extracellular adenosine signaling in tissue adaptation and wound healing. Therefore, we hypothesized a functional contribution for the endogenous production of adenosine during ALI resolution. As a model, we administered intratracheal LPS and observed peak lung injury at 3 d, with resolution by d 14. Treatment with pegylated adenosine‐deaminase to enhance extracellular adenosine breakdown revealed impaired ALI resolution. Similarly, genetic deletion of cd73, the pacemaker for extracellular adenosine generation, was associated with increased mortality (0% wild‐type and 40% in cd73–/– mice; P<0.05) and failure to resolve ALI adequately. Studies of inflammatory cell trafficking into the lungs during ALI resolution revealed that regulatory T cells (Tregs) express the highest levels of CD73. While Treg numbers in cd73–/– mice were similar to controls, cd73‐deficient Tregs had attenuated immunosuppressive functions. Moreover, adoptive transfer of cd73‐deficient Tregs into Rag–/– mice emulated the observed phenotype in cd73–/– mice, while transfer of wild‐type Tregs was associated with normal ALI resolution. Together, these studies implicate CD73‐dependent adenosine generation in Tregs in promoting ALI resolution.—Ehrentraut, H., Clambey, E. T., McNamee, E. N., Brodsky, K. S., Ehrentraut, S. F., Poth, J. M., Riegel, A. K., Westrich, J. A., Colgan, S. P., Eltzschig, H. K. CD73+ regulatory T cells contribute to adenosine‐mediated resolution of acute lung injury. FASEB J. 27, 2207–2219 (2013). www.fasebj.org

The toll-like receptor 4-antagonist eritoran reduces murine cardiac hypertrophy.

Toll‐like receptor 4 (TLR4) recognizes lipopolysaccharides and endogenous ligands released after organ injury. Deficiency of TLR4 attenuates the development of left ventricular hypertrophy after transverse aortic constriction (TAC) in mice. We hypothesized that application of the TLR4 antagonist eritoran may also reduce cardiac hypertrophy after TAC surgery.

IN VIVO TOLL-LIKE RECEPTOR 4 ANTAGONISM RESTORES CARDIAC FUNCTION DURING ENDOTOXEMIA

ABSTRACT Severe sepsis and septic shock are often accompanied by acute cardiovascular depression. Lipopolysaccharide (LPS) signaling via Toll-like receptor 4 (TLR4) can induce septic organ dysfunction. The aim of this study was to elucidate the in vivo impact of pharmacological TLR4 antagonism on LPS-induced cardiovascular depression using eritoran tetrasodium (E5564). To simulate sepsis, C3H/HeN mice were challenged i.p. with 2 mg/kg body weight LPS. With the intent to antagonize the LPS effects, eritoran was administered i.v. (4 mg/kg body weight). Physical activity, peripheral blood pressure, and heart frequency were recorded before and after LPS and eritoran injection. In addition, intracardiac hemodynamic parameters were analyzed with a pressure conductance catheter. After 2 and 6 h of LPS stimulation ± eritoran treatment, the hearts and aortae were harvested, and TLR as well as inflammatory mediator expression was measured using reverse transcription–quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Lipopolysaccharide significantly decreased arterial blood pressure over time. Administration of eritoran partially prevented the LPS-dependent reduction in blood pressure and preserved cardiac function. In addition, LPS increased the expression of CD14 and TLR2 in cardiac and aortic tissue. In aortic tissue, eritoran attenuated this increase, whereas no significant reduction was observed in the heart. Furthermore, cardiac and aortic inducible nitric oxide synthetase mRNA levels were significantly increased 6 h after LPS application. This effect was reduced in the presence of eritoran. In summary, the beneficial influence of eritoran on cardiovascular function in vivo seems to rely mainly on reduction of LPS-induced inducible nitric oxide synthetase expression as well as on attenuated cytokine expression in the vascular wall.

Vascular Dysfunction following Polymicrobial Sepsis: Role of Pattern Recognition Receptors

Aims Aim was to elucidate the specific role of pattern recognition receptors in vascular dysfunction during polymicrobial sepsis (colon ascendens stent peritonitis, CASP). Methods and Results Vascular contractility of C57BL/6 (wildtype) mice and mice deficient for Toll-like receptor 2/4/9 (TLR2-D, TLR4-D, TLR9-D) or CD14 (CD14-D) was measured 18 h following CASP. mRNA expression of pro- (Tumor Necrosis Factor-α (TNFα), Interleukin (IL)-1β, IL-6) and anti-inflammatory cytokines (IL-10) and of vascular inducible NO-Synthase (iNOS) was determined using RT-qPCR. Wildtype mice exhibited a significant loss of vascular contractility after CASP. This was aggravated in TLR2-D mice, blunted in TLR4-D animals and abolished in TLR9-D and CD14-D animals. TNF-α expression was significantly up-regulated after CASP in wildtype and TLR2-D animals, but not in mice deficient for TLR4, -9 or CD14. iNOS was significantly up-regulated in TLR2-D animals only. TLR2-D animals showed significantly higher levels of TLR4, -9 and CD14. Application of H154-ODN, a TLR9 antagonist, attenuated CASP-induced cytokine release and vascular dysfunction in wildtype mice. Conclusions Within our model, CD14 and TLR9 play a decisive role for the development of vascular dysfunction and thus can be effectively antagonized using H154-ODN. TLR2-D animals are more prone to polymicrobial sepsis, presumably due to up-regulation of TLR4, 9 and CD14.

Tlr4 Deficiency Protects against Cardiac Pressure Overload Induced Hyperinflammation.

Transverse aortic constriction provokes a pro-inflammatory reaction and results in cardiac hypertrophy. Endogenous ligands contribute to cardiac hypertrophy via toll-like receptor (TLR)-4 binding. A lack of TLR4 signaling diminishes hypertrophy and inflammation. Wild type mice undergoing aortic constriction respond to a lipopolysaccharide second-hit stimulus with hyperinflammation. The objective of this study was to assess whether other second-hit challenges utilizing TLR ligands provoke a comparable inflammatory reaction, and to find out whether this response is absent in TLR4 deficient mice. Assuming that cardiac stress alters the expression of pattern recognition receptors we analyzed the effects of transverse aortic constriction and second-hit virulence factor treatment on TLR expression, as well as cytokine regulation. Wild type and Tlr4 -/- mice were subjected to three days of TAC and subsequently confronted with gram-positive TLR2 ligand lipoteichoic acid (LTA, 15mg/g bodyweight) or synthetic CpG-oligodesoxynucleotide 1668 thioate (20 nmol/kg bodyweight, 30 min after D-galactosamin desensitization) signaling via TLR9. Hemodynamic measurements and organ preservation were performed 6 h after stimulation. Indeed, the study revealed a robust enhancement of LTA induced pattern recognition receptor and cytokine mRNA expression and a LTA-dependent reduction of hemodynamic pressure in TAC wild type mice. Second-Hit treatment with CpG-ODNs led to similar results. However, second-hit effects were abolished in Tlr4 -/- mice. In total, these data indicate for the first time that cardiac stress increases the inflammatory response towards both, gram-negative and gram-positive, TLR ligands as well as bacterial DNA. The decrease of the inflammatory response upon TLR2 and -9 ligand challenge in TAC Tlr4 -/- mice demonstrates that a lack of TLR4 signaling does not only prevent left ventricular hypertrophy but also protects the mice from a cardiac stress induced hyperinflammatory reaction.

Experimental murine acute lung injury induces increase of pulmonary TIE2-expressing macrophages

BackgroundBreakdown of the alveolo-capillary wall is pathognomonic for Acute Lung Injury (ALI). Angiopoietins, vascular-specific growth factors, are linked to endothelial barrier dysfunction, and elevated Angiopoietin-2 (ANG2) levels are associated with poor outcome of ALI patients. Specialized immune cells, referred to as ‘TIE2-expressing monocytes and macrophages’ (TEM), were shown to specifically respond to ANG2 binding. However, their involvement in acute inflammatory processes is so far completely undescribed. Thus, our aim was to assess the dynamics of TEMs in a murine model of ALI.ResultsIntratracheal instillation of LPS induced a robust pulmonary pro-inflammatory response with endothelial barrier dysfunction and significantly enhanced ANG2 expression. The percentage number of TEMs, assessed by FACS analysis, was more than trebled compared to controls, with TEM count in lungs reaching more than 40% of all macrophages. Such distinct dynamic was absent in all other analyzed compartments (alveolar space, spleen, blood). Incubation of the monocytic cell line THP-1 with LPS or TNF-α resulted in a dose-dependent, significant upregulation of TIE2, suggesting that not recruitment from extra-pulmonary compartments but TIE2 upregulation in resident macrophages accounts for increased lung TEM frequencies.ConclusionsFor the first time, our data provide evidence that the activity of TEMs changes at sites of acute inflammation.