during the course of sepsis pattern recognition receptors ( prrs ) of the innate immune system such as toll - like receptors ( tlrs ) have been shown to play a decisive role in the disease process .
toll - like receptors bind pathogen associated molecular patterns ( pamps ) originating from non - self - sources as well as damage associated molecular patterns ( damps ) deriving from the host organism itself . in the human organism eleven tlrs
are expressed , among those tlr4 the receptor for endotoxins has been described most extensively . however , in the last few years another tlr has entered the focus of scientific interest : the receptor for viral and bacterial dna tlr9 .
bacterial dna is characterized by unmethylated cytosine - phosphate - guanine oligodesoxynucleotide ( cpg motifs ) , which are much less prevalent in mammalian dna .
this receptor has been found to be of pivotal importance during polymicrobial sepsis [ 3 , 4 ] .
the reason for its relevance may lie in the common appearance of cpg motifs in all bacterial and viral dna . therefore , tlr9 can be regarded as a central receptor for inflammatory response in any kind of sepsis .
importantly , the presence of impaired cardiovascular function in sepsis is associated with significantly increased mortality [ 5 , 6 ] .
hence , any successful intervention should aim to improve sepsis - dependent suppression of cardiovascular function . 
 in vitro tlr9
signaling induced by the application of the synthetic stimulatory oligonucleotide 1668-thioate suppressed cardiac contractility via increased inos expression , which could be antagonized by s - methylisothiourea ( smt ) . in addition , myocytes with tlr9-deficiency ( tlr9-d ) proved to be insensitive to stimulation with a synthetic substitute for bacterial dna .
thus , pharmacological antagonism of tlr9 might protect the cardiovascular system from the deleterious effects of bacterial dna .
dilatation of blood vessels exposed to polymicrobial stimuli can be prevented by the synthetic odn ( h154-thioate ) inhibiting tlr9 .
however , the in vivo effect of tlr9 antagonism on the heart has not yet been investigated .
therefore , we wondered whether application of a synthetic oligodesoxynucleotide ( 1668-thioate ) would be sufficient to depress cardiac function in vivo . with respect to the importance of effective inhibition of tlr signaling in sepsis , the aim of the present study was to test different tlr9 inhibitors ( h154-thioate , irs954-thioate , and chloroquine ) for their effectiveness in protecting the cardiovascular system during systemic inflammation . in order to distinguish tlr9 signaling from other inflammatory pathways the specific tlr9 ligand 1668-thioate was chosen as stimulus .
ten - to - twelve - week - old male c57bl/6 mice were purchased from charles river , sulzfeld / germany .
the body weight varied from 20 to 25  g. tlr9-deficient mice ( tlr9-d ; backcrossed on c57bl/6 background ) were kindly provided by professor shizuo akira ( department of host defense , research institute for microbial diseases , osaka university , japan ) .
all animal studies were approved by the state office for nature , environment and consumer affairs ( landesamt fr natur , umwelt und verbraucherschutz ) of north - rhine westphalia ( recklinghausen , germany ) and conformed to the guidelines for animal experimentation of the national institute of health ( nih publication no .
buprenorphine was administered ( 0.1  mg / kg subcutaneously ) . 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 10 bacteria / ml ) .
thereafter , tnf- protein was measured by elisa in the supernatant of the cell culture .
stimulation led to a 30-fold increase of tnf- 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 [ 810 ] .
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- 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 ) ) .
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 .
all animals were initially treated with d - galactosaminen ( d - galn ; 1  g / kg bw , roth , karlsruhe , germany ) .
d - galactosaminen was applied to slow down the hepatic degradation of cpg - odns [ 11 , 12 ] . in control experiments
d - galn alone did not induce an inflammatory response . in order to induce a systemic inflammation
thirty minutes later all mice were injected i.p . with 1668-thioate ( 5-tccatgacgttcctgatgct ; tibmolbiol , berlin , germany ; 2  nmol / g bw = 12.1  mg / kg bw ) in a total volume of 400  l pbs or with the same amount of pbs .
another 30 minutes later the mice received intravenous ( i.v ) treatment with one of four agents : ( 1 ) a control odn ( 1612-thioate ; 5-gctagatgttagcgt-3 ; tibmolbiol , berlin , germany ; 2  nmol / g bw = 9.3  mg / kg bw ) , ( 2 ) with h154-thioate ( 5-cctcaagcttgagggg-3 ; tibmolbiol , berlin , germany ; 8  mg / kg bw ) , ( 3 ) with irs954-thioate ( 5-tgctcctggaggggttgt-3 ; tibmolbiol , berlin , germany ; 8  mg / kg bw ) , ( 4 ) with chloroquine ( 10  mg / kg bw ; bayer vital gmbh , leverkusen , germany ) .
the mrna expression levels of tnf- , il-6 , and il-1 were determined using taqman real - time quantitative pcr ( rt - qpcr , applied biosystems , darmstadt , germany ) . upon excision of the hearts
total rna was isolated ( trizol , applied biosystems ) and first - strand cdna was synthesized using the high - capacity cdna transcription kit ( applied biosystems ) with random hexameric primers according to the manufacturer 's protocol .
rt - qpcr was performed and analyzed with cdna ( diluted 1  :  10 ) on an abi prism 7900 sequence detection system and sds2.2 software ( applied biosystems ) .
target gene expression was normalized to an internal control ( glyceraldehyde-3-phosphate dehydrogenase , gapdh ) .
relative rt - pcr was performed using taqman gene expression master mix ( part 4369016 ; applied biosystems ) with the following primers : gapdh ( mm99999915_g1 ) , tnf- ( mm00443258_m1 ) , il-1 ( mm99999061_g1 ) , and il-6 ( mm01210732_g1 ) .
all murine primers were measured using fam tamra chemistry and the relative standard curve method . at the end of rt - qpcr cycle
six hours after stimulation with 1668-thioate hemodynamic parameters which included left ventricular systolic pressure ( lvsp ) , stroke volume ( sv ) , left ventricular end - diastolic pressure ( lvedp ) , cardiac output ( co ) , and contractility indices ( dp / dtmax and dp / dtmin ) were recorded using a pressure - volume catheter according to the manufacturer 's manual ( millar instruments , houston tx ) .
additionally , body temperature was monitored in representative mice using a rectal probe ( figure 2(a ) ) . for detailed descriptions
significance testing included one - way anova followed by newman - keuls post hoc analysis .
clinical appearance as well as body temperature was investigated in wt and tlr9-d mice up to 18  h after 1668-thioate stimulation .
in addition , survival was monitored in all groups of stimulated wt mice . after only 2  h wt mice started to display sepsis - like symptoms such as ruffled fur , food refusal , and limited ability to respond to external stimuli . in the following
neither the specific tlr9 antagonist irs954 nor the unspecific tlr9 inhibitor chloroquine improved the clinical appearance of wt mice stimulated with 1668-thioate in vivo .
however , the application of h154-thioate improved clinical behavioral patterns obviously : none of the mice in this group showed external signs of systemic inflammation . in accordance with this tlr9-d mice also did not exhibit any clinical symptoms .
six hours after 1668-thioate stimulation body temperature in both wt and tlr9-d mice significantly decreased ; however , body temperature in tlr9-d mice remained above that of wt mice ( figure 2(a ) ) .
interestingly , wt mice cotreated with h154-thioate presented nearly the same temperature as tlr9-d mice ( figure 2(a ) ) .
these findings concur with the observation that all wt animals treated with 1668-thioate were dead shortly after 6 hours .
h154-thioate proved to be the most effective substance improving survival by 40% ( figure 2(b ) ) .
furthermore , all tlr9-d animals survived longer than 18 hours after 1668-thioate application ( data not included in figure 2(b ) ) .
two hours and six hours after 1668-thioate stimulation mediators of inflammation ( tnf- , il-1 , 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-1 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- nor il-1 ( figures 3(b ) and 3(d ) ) .
neither irs954-thioate nor chloroquine suppressed cardiac tnf- or il-1 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
parameters of cardiovascular performance monitored with a pressure - volume catheter in wt as well as in tlr9-d mice 6  h after stimulation with 1668-thioate are given in figures 4(a)4(f ) .
there was a significant impairment of all measured functional parameters in stimulated wt mice compared to pbs controls . in agreement with cytokine mrna expression , additional application of h154-thioate significantly improved lv function
heart rate ( hr ; data not shown ) , left ventricular systolic pressure ( lvsp ) , stroke volume ( sv ) , cardiac output ( co ) , end - diastolic volume ( edv ) , velocity of pressure increase ( dp / dtmax ) , and velocity of pressure decrease ( dp / dtmin ) were improved by h154-thioate in comparison to 1668-thioate + pbs ( figures 4(a)4(f ) ) .
deficiency for tlr9 entirely prevented any deterioration of cardiac function ( figures 4(a)4(f ) ) .
this study aimed to extend the in vitro finding that tlr9 stimulation decreases cardiomyocyte contractility to the in vivo setting . to our knowledge
the present results are first to formally demonstrate that specific tlr9 stimulation with 1668-thioate lowers hemodynamic parameters in vivo in a murine model of systemic inflammation .
the second attempt of the present investigation was to test possible tlr9 inhibitors ( h154-thioate , irs954-thioate , and chloroquine ) for their cardioprotective properties .
the three tested inhibitors exhibited differential potencies ; that is , h154-thioate proved to be the most effective substance .
1668-thioate has been applied in various experimental settings for the induction of a tlr9-dependent systemic inflammation as well as organ dysfunction [ 7 , 11 , 14 , 16 , 17 ] .
the effect of tlr9-dependent systemic inflammation as well as organ dysfunction seems to be dose dependent as a low concentration ( 0.25  nmol / g ) can serve as a mild stimulus for cardiac preconditioning , whereas higher concentrations in the range from 0.5 to 1  nmol / g induce sepsis - like inflammation [ 7 , 17 ] . in order to warrant clear changes in cardiac function the formerly applied dose of 1 
nmol / g was doubled to 2  nmol / g bw in the current study .
furthermore , the relatively high dose of 1668-thioate applied here seemed to be meaningful with respect to testing antagonizing strategies .  
treatment either with a control odn ( 1612-thioate ) or with one of the three inhibitors .
application of the antagonists was to avoid a possible direct interaction between stimulating and inhibitory substances in the peritoneal cavity .
specific tlr9 stimulation led to a time - dependent upregulation of cardiac tnf- , il-1 , and il-6 mrna expression with a peak 2  h after stimulation and a reduced level 4  h later .
this time course as well as the tlr9-dependent cytokine induction confirms earlier findings of our group .
an interesting new aspect is that costimulation with 1612-thioate increased the expression of all three pro - inflammatory mediators at 2  h , reaching significance in the case of il-1 and il-6 mrna .
however , prestimulation with 1668-thioate may sensitize the organism to succeeding stimuli in a way that originally inert substances ( 1612-thioate ) are able to act as second hits , thereby further increasing the inflammatory response .
a possible explanation for the enhanced mediator response may lie in the weak immune stimulatory properties of the charged phosphorothioate backbone of 1612-thioate .
h154-thioate was the sole antagonist , which significantly depressed the upregulation of the three tested cytokines 2  h after stimulation with 1668-thioate .
chloroquine failed to develop any protective influence at this time point , but even enhanced the expression of il-6 ( figures 3(a ) , 3(c ) , and 3(e ) ) .
four hours later , the expression of the three cytokines had fallen to at least 60% , which was still significantly above the pbs and tlr9-d controls .
also in the groups with additional application of antagonists the absolute levels of cytokine expression had fallen .
significant antagonistic effects appeared only for the combinations of il-6 and h154-thioate as well as il-6 and chloroquine ( figures 3(b ) , 3(d ) , and 3(f ) ) .
ligands applied from the extracellular space have to be internalized and transported to the endosomal compartment .
chloroquine has been shown to disrupt this vesicle trafficking and acidification , which can explain the inhibition of the innate immune response .
these data were mainly derived from in vitro experiments . however , there are reports that chloroquine is also effective in vivo [ 8 , 20 ] . in a model of polymicrobial sepsis , chloroquine improved survival and reduced renal injury as well as systemic inflammation .
with respect to survival these findings appear to contradict ours . however , there are differences between the model in this study and that of yasuda et al . .
we applied a specific tlr9 stimulus in a concentration high enough to induce a drastic systemic inflammation causing death of 100% of the animals shortly after 6  h , whereas yasuda et al .
applied cecal ligation and puncture ( clp ) with a mortality of 50% after more than 48  h ; that is , the clp - dependent inflammation was less severe than the one in the present study .
in addition to the inhibition of vesicle trafficking , it has been proposed that chloroquine decreases tlr9 protein .
thus , tlr9 signaling itself may be a major target for the protective actions of chloroquine .
this downregulation of tlr9 protein was detected 18  h after application of chloroquine ; this mechanism is possibly too slow to develop a beneficial influence in our model . in another attempt , hong et al
. demonstrated that chloroquine at a dose of 30  mg / kg bw could protect mice from lethal challenge by 1668-thioate , whereas chloroquine at a dose of 25  mg / kg bw could decrease serum tnf- and il-6 in rats injected with sublethal doses of cpg - odn . in our hands , preliminary tests of a high chloroquine dose of 30  mg / kg bw caused a 100% mortality immediately after i.v .
we therefore stayed with a dose of 10  mg / kg bw as explained in material and methods section .
this divergence in chloroquine sensitivity may be attributed to differences in mouse strains ( balb / c versus c57bl/6n mice ) . taken together , in our model of cpg - induced sepsis the protective abilities of chloroquine could not be confirmed .
synthetic oligonucleotides containing inhibitory properties such as h154-thioate and irs954-thioate are frequently used in experimental studies to suppress tlr signaling [ 21 , 22 ] .
pisetsky 's group described synthetic oligonucleotides containing poly - g sequences , which block bacterial dna - induced activation [ 23 , 24 ] .
furthermore , it has been suggested that suppressive odns interfere with the phosphorylation of signal transducer and activator of transcriptions 1 and 4 ( stat1 and stat4 , resp . ) , thereby blocking inflammation mediated by stat - associated signaling cascades .
this interaction may be highly specific because suppressive odns do not bind to other molecules in the nfb and mapk regulatory cascade , and control odns do not bind to stat1 or stat4 [ 25 , 26 ] .
furthermore , in vitro studies have demonstrated that inhibitory odns preferentially bind to the c - terminal of tlr9 , competing for cpg - odn binding . with respect to the structural details of inhibitory odns ,
ashman et al . demonstrated in vitro that a specific motif ( ccx(not - c)(not - c)xxggg ( x = any base ) ) provides the sequences required to block tlr9 in human b cells and hek cells transfected with human tlr9 . extending the sequence by four to five bases at the 5 end enhanced activity , even more so when a phosphorothioate backbone replaced the native phosphodiester backbone   ( for further details , see [ 25 , 26 ] ) .
in addition to the molecular composition of an inhibitory odn also the targeted cell type has been shown to influence the inhibitory potency of the respective odn ; that is , some inhibitory sequences that function well in macrophages have been shown to be inefficient in b cells . taken together ,
the workings of molecular blocking mechanisms of inhibiting odn have not yet been unraveled ; differences in the potency of h154-thioate and irs954-thioate up to now can not be attributed to their molecular structure .
are generated in a highly controlled environment , which is not the case in living organisms .
therefore , transfer from in vitro to in vivo can not be assumed to be self - evident .
thus , the presented cardiodepressive effect of 1668-thioate stimulation in vivo enhances our knowledge of the understanding of tlr9 stimulation .
this is further supported by the total insensitivity of tlr9-d mice to this stimulation . to our knowledge , this study is the first demonstration that specific tlr9 stimulation with 1668-thioate lowers hemodynamic parameters in vivo in a murine model of systemic inflammation . in our experimental setting h154-thioate
there was a significant impairment of all measured functional parameters in stimulated wt mice compared to pbs controls . in agreement with cytokine mrna expression , additional application of h154-thioate significantly improved lv function when compared to mice treated with 1668-thioate + pbs .
specifically , heart rate , left ventricular systolic pressure , stroke volume , cardiac output , end - diastolic volume , velocity of pressure increase , and velocity of pressure decrease were improved by h154-thioate in comparison to 1668-thioate + pbs ( figures 4(a)4(f ) ) .
deficiency for tlr9 entirely prevented any deterioration of cardiac function ( figures 4(a)4(f ) ) .
our finding that h154-thioate protected the heart against tlr9-stimulation complements the earlier observation that h154-thioate prevented tlr9-dependent vascular relaxation .
the importance of tlr9 signaling in polymicrobial infection has already been shown by others and by our group [ 3 , 4 ] . a possible reason for this may lie in the ability of tlr9 to bind the dna of gram - positive as well as gram - negative bacteria and thus to react to a broad range of stimuli [ 3 , 30 , 31 ] .
this may have contributed to the disappointing results of the application of eritoran ( e5564 ) , a tlr4 antagonist , evaluated in the access ( a controlled comparison of eritoran and placebo in patients with severe sepsis ) trial , a global , randomized , double - blind , placebo - controlled phase iii study .
taken together , here we show that systemic tlr9 stimulation is able to depress cardiac function in vivo and that a pharmacological intervention is possible and may be a promising strategy for human clinical trials in future .
 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 . 