Toll-Like Receptor 2 Mediates Staphylococcus aureus–Induced Myocardial Dysfunction and Cytokine Production in the Heart
Background— Staphylococcus aureus sepsis is associated with significant myocardial dysfunction. Toll-like receptor 2 (TLR2) mediates the inflammatory response to S aureus and may trigger an innate immune response in the heart. We hypothesized that a TLR2 deficiency would attenuate S aureus–induced cardiac proinflammatory mediator production and the development of cardiac dysfunction.
Methods and Results— Wild-type and TLR2-deficient (TLR2D) mice were studied. S aureus challenge significantly increased tumor necrosis factor, interleukin-1β, and nitric oxide expression in hearts of wild-type mice. This response was significantly blunted in TLR2D mice. Hearts from TLR2D mice had impaired S aureus–induced activation of interleukin-1 receptor–associated kinase, c-Jun NH2 terminal kinase, nuclear factor-κB, and activator protein-1. Moreover, hearts from TLR2D mice were protected against S aureus–induced contractile dysfunction.
Conclusions— These results show for the first time that TLR2 signaling contributes to the loss of myocardial contractility and cytokine production in the heart during S aureus sepsis.
Received June 17, 2004; revision received July 12, 2004; accepted August 2, 2004.
Sepsis develops when the initial appropriate response to an infection becomes amplified and then dysregulated. Myocardial dysfunction, a central component in the complex pathophysiology of sepsis, contributes to the high mortality associated with this disorder. Cardiac dysfunction starts during the first 24 hours of the development of sepsis, and it is reversible in survivors within 7 to 10 days.1 Experimental studies indicate that the pattern of cardiac dysfunction observed in Gram-negative sepsis is also encountered in Gram-positive sepsis.2 Supporting these finding are clinical data showing that ejection fraction recorded in humans with Gram-positive or Gram-negative sepsis does not differ significantly.3,4 Although these studies suggest that Gram-positive and Gram-negative bacteria activate analogous signaling pathways in the heart, progress in the treatment of sepsis-induced myocardial depression has been hampered by a poor understanding of the molecular mechanisms involved in this condition.
Increased expression of tumor necrosis factor (TNF) and interleukin-1β (IL-1β) occurs in the heart in experimental sepsis.5,6 There is now consensus that these cytokines contribute to cardiac decompensation in human sepsis.7,8 However, the proximal events that trigger and sustain their expression in the heart are not well understood. We previously reported that toll-like receptor-4 (TLR4) and CD14 mediate the induction of cardiac TNF, IL-1β, and nitric oxide (NO) after lipopolysaccharide (LPS) administration.9,10 More important, TLR4- and CD14-deficient mice are protected against LPS-induced left ventricular (LV) dysfunction.10,11 Although these studies provided new insights into the pathogenesis of LPS-induced cardiac depression, the mechanisms through which severe staphylococcal sepsis can mimic Gram-negative sepsis remain undefined.
The discovery of mammalian TLRs has provided an explanation for how innate immune cells recognize microbial antigens.12 Evidence now exists that TLR2 is essential for S aureus recognition by immune cells. Yoshimura et al13 were the first to report that S aureus induced cytokine production via TLR2. Subsequently, TLR2-deficient (TLR2D) mice were shown to produce reduced levels of cytokines in response to heat-killed S aureus and peptidoglycan.14,15 Although studies have primarily addressed the role of these receptors in immune cells, TLRs are expressed in the heart, where they may also serve as proinflammatory receptors.16–18 Thus, the development of LV dysfunction during severe staphylococcal sepsis may involve excessive TLR activation within the cardiac compartment. Accordingly, in this study, we determined whether TLR2 played a role in S aureus–induced cardiac inflammation and LV dysfunction.
TLR2D mice were generated as described previously.14 Homozygous mice were interbred, and age- and sex-matched mice were used. The Institutional Animal Care and Use Committee approved all experiments.
S aureus isolated from a child with septic shock was frozen at −70°C in trypticase soy broth with 10% glycerol (pH 7.3). Bacterial pellets were washed with pyrogen-free phosphate-buffered saline (Invitrogen) and resuspended to a concentration of 5 to 25×108 colony-forming units (CFU)/mL. Bacterial counts were determined by serial-dilution plating.
Heat-inactivated S aureus was prepared from mid–log-phase cultures, resuspended at a density of 1010 CFU/mL, and heat inactivated (30 minutes, 80°C). Endotoxin was not detectable by a Limulus assay with a sensitivity of <0.5 endotoxin units (EU)/mL (BioWhittaker).
Animal Inoculation and Organ Harvest
Mice were inoculated intraperitoneally with 1 to 5×108 CFU of S aureus. All mice were killed with a lethal injection of sodium pentobarbital (80 mg/kg). Hearts were harvested at 0, 2, 4, 6, and 12 hours after S aureus challenge for determination of TNF and IL-1β gene and protein expression. For determination of NO synthase-2 (NOS2) protein expression, nitrite production, and cGMP production, animals were humanely killed at 0, 6, and 12 hours after infection. Hearts were harvested at 0, 2, 4, and 6 hours for assessment of nuclear factor (NF)-κB, activator protein (AP)-1, interleukin-1 associated kinase (IRAK), and c-Jun NH2 terminal kinase (JNK) activation.
Cloning of the Murine TLR2 cDNA
A full-length murine sequence for TLR2 from the NCBI expressed-sequence-tag database was used to design primers (AF124741). The TLR2 sense primer (5′CTTGCTCGTAGGTGCC-3′) was derived from bases 2097 to 2112 of the coding region, and the antisense primer (5′CACCACTCGCTCCGTA-3′) was derived from bases 2414 to 2429.
TLR2 Expression in Myocardial Tissue
TLR2 mRNA was detected by ribonuclease protection assay (RPA), as previously described.9 TLR2 protein was detected by Western blot analysis with use of a mouse TLR2 polyclonal antibody (1:200, eBioscience). TLR2 protein expression was normalized to β-actin, and fold increase was calculated versus the time 0-hour value set at 1.
TNF and IL-1β RPA
TNF and IL-1β gene expression was determined with a multiprobe RPA system (RiboQuant, Pharmingen) as previously described.9 Signals were normalized to ribosomal protein L32 mRNA (L32) and quantified with Image Quant software (Molecular Dynamics). Expression of L32 is not affected in different models of cardiac injury.10,19
Myocardial TNF and IL-1β ELISA
Hearts were harvested at specific times, and homogenates were prepared as previously described.9 TNF and IL-1β proteins were measured by ELISA (R&D Systems). Data were expressed as picograms per milligram protein.
NO Synthase-2 Western Blot Analysis
Protein (100 μg/lane) was separated on 8% sodium dodecyl sulfate polyacrylamide gels. The membrane was immunoblotted with a rabbit anti-NOS2 antibody (1:500, Sigma). NOS2 protein expression was normalized to β-actin expression. Fold increase was calculated versus the time 0-hour value set at 1.
Measurement of Nitrite and cGMP Concentration
Cardiac NO production was measured with a nitrate/nitrite colorimetric kit (Cayman Chemical). The NO concentrations were expressed in nanomoles per milligram protein. For measurement of cGMP, hearts were homogenized as previously described.10 cGMP was measured by radioimmunoassay (Biomedical Technologies Inc). Data were calculated as picomoles cGMP per milligram of trichloroacetic acid–precipitated protein. Fold increase was calculated versus the time 0-hour value set at 1.
Assay of IRAK and JNK Activity
Cytoplasmic proteins (500 μg) were immunoprecipitated with 5 μg IRAK-1 antibody (Santa Cruz) at 4°C overnight. After addition of 25 μL protein A–agarose beads (Santa Cruz) for 2 hours at 4°C, the precipitates were collected by centrifugation. The pellets were washed twice in lysis buffer and twice in kinase buffer (25 mmol/L Tris [pH 7.5], 5 mmol/L β-glycerophosphate, 2 mmol/L dithiothreitol, 0.1 mmol/L sodium orthovanadate, 10 mmol/L MgCl2, and 0.5% Triton X-100). Kinase reactions were performed in kinase buffer containing myelin basic protein (2 μg), 10 μmol/L ATP, and 5 μCi [γ-32P]ATP (37°C, 30 minutes). Densitometric analysis was performed with Image Quant (Molecular Dynamics).
JNK activity was determined by phosphorylation of glutathione S-transferase–c-Jun (Biomol Research). Nuclear extracts were prepared as previously described, and 50 μg was immunoprecipitated with 5 μg JNK1/2 antibody (Santa Cruz).9 Kinase assay conditions were as described earlier, except that glutathione S-transferase–c-Jun (5 μg) was used as the substrate.
Myocardial Nuclear Extraction and Electrophoretic Mobility Shift Assays
NF-κB and AP-1 electrophoretic mobility shift assays were carried out as previously described.9 For NF-κB supershift assays, the nuclear extracts were incubated with polyclonal anti-p50 or anti-p65 antibodies (Geneka Biotechnology).
Isolated-Heart Perfusion and Measurement of LV Function
Mice were injected intraperitoneally with heparin (10 000 U/kg, Sigma) and anesthetized with 2,2,2-tribromoethanol (16 μL/g of a 2.5% solution). A short perfusion cannula was inserted into the aortic root, and retrograde perfusion was initiated immediately at 80 mm Hg with modified Krebs-Henseleit buffer (118 mmol NaCl, 24 mmol NaHCO3, 4.7 mmol KCl, 1.2 mmol KH2PO4, 1.2 mmol MgSO4, 2.2 mmol CaCl2, 10 mmol glucose, and 2 mmol pyruvate; pH 7.4, 37°C). The perfusate was gassed with 95% O2 and 5% CO2. A polyvinyl chloride balloon was inserted into the LV through an incision in the left atrium and was connected to a pressure transducer (ML844, AD Instruments). The balloon was filled with water to adjust the LV end-diastolic pressure to 7 to 10 mm Hg. Hearts were paced with a Grass SD9 stimulator (Grass Instruments) at 420 beats per minute. Functional data were recorded at 1 kHz on a data acquisition system (PowerLab, AD Instruments). LV developed pressure was calculated as the difference between peak systolic pressure and LV end-diastolic pressure. All glassware was baked at 190°C for 4 hours.
Contractile function was assessed in 4 groups. Groups 1 and 2 consisted of buffer-perfused hearts from wild-type (n=3) and TLR2D (n=3) mice, respectively. Groups 3 and 4 consisted of S aureus–challenged hearts from wild-type (n=5) and TLR2D (n=5) mice, respectively. After stabilization (30 minutes), the time was set to 0, and heat-killed S aureus was admixed with the perfusate (108 CFU/mL, groups 3 and 4). LV developed pressure was monitored for 80 minutes. At the end of each experiment, hearts were frozen and later used for determination of TNF and IL-1β protein by ELISA.
Values were expressed as mean±SEM. ANOVA followed by Fisher’s protected least significant difference test (where appropriate) was used to determine significant differences in S aureus–induced proinflammatory mediator production, IRAK/NF-κB activation, and LV dysfunction between wild-type and TLR2D mice. Baseline physiological characteristics between hearts from wild-type and TLR2D mice were compared by unpaired t test. A probability value ≤0.05 was considered significant.
Clinical Manifestations of Infection
Eight hours after challenge, both wild-type and TLR2D mice developed signs of sepsis (ruffled hair and diarrhea). The intensity of bacteremia was similar in both groups at 6 hours (7.8×104±3.3×103 versus 8.1×104±3.5×103 colonies/mL blood) and 12 hours (2×106±9.5×105 versus 3.4×106±1×106 colonies/mL blood) after inoculation. During the 12-hour period there were no deaths.
S aureus Infection Induces TLR2 Expression in the Heart
TLR2 mRNA was expressed at low levels in the naive heart. Cardiac TLR2 gene expression increased significantly (P≤0.05) in S aureus–challenged wild-type mice at 2 (101%), 4 (208%), and 6 hours (169%, Figure 1A and 1B). TLR2 protein expression increased by 28% (2 to 4 hours, Figure 1C and 1D) but decreased to almost baseline by 6 hours. These studies document that TLR2 is upregulated in the heart during S aureus infection.
S aureus–Induced Production of TNF and IL-1β Is Modulated in the Absence of TLR2
TNF and 1L-1β gene and protein expressions were measured in wild-type and TLR2D mice after S aureus infection. S aureus induced a significant increase (4 and 6 hours, P≤0.05) in TNF and IL-1β mRNA transcripts (Figure 2A and 2B) in the hearts of wild-type mice. Although S aureus also induced TNF and IL-1β mRNA in the hearts of TLR2D mice, the magnitude of the response was significantly attenuated.
Figure 3A and 3B shows that the kinetics of TNF and IL-1β protein production paralleled the upregulation of TNF and IL-1β mRNA after S aureus challenge. Myocardial TNF protein levels were significantly higher (P≤0.05) at 4 and 6 hours after S aureus infection in wild-type mice. Peak TNF production in vivo was ≈20-fold higher (4.2 versus 0.2 pg/mg, P≤0.05) in wild-type mice compared with TLR2D mice. Tissue levels of IL-1β also were significantly higher in wild-type mice.
NO and cGMP Production Is Attenuated in S aureus–Infected TLR2D Mice
At baseline, extracts from both wild-type and TLR2D mice had minimally detectable NOS2 protein (Figure 4A). After S aureus challenge, cardiac NOS2 expression was increased significantly (P≤0.05) at 6 hours in wild-type mice, whereas minimal change was detected in TLR2D mice (Figure 4B). By 12 hours, no differences were noted in NOS2 expression. As shown in Figure 5C and 5D, myocardial NO and cGMP levels were increased significantly (P≤0.05) at 6 hours after S aureus challenge in wild-type mice only.
Altered Intracellular Signaling in the Hearts of S aureus–Infected TLR2D Mice
To document TLR2 signaling in the heart, we measured S aureus–induced activation of IRAK, a central component in TLR2 signaling.20 IRAK activity was significantly blunted (Figure 5A and 5B) in the hearts of TLR2D mice. In agreement with IRAK activation, a significant increase in myocardial NF-κB DNA binding activity was observed in wild-type mice (n=4 animals per time point) within 4 hours of S aureus challenge (Figure 5C and 5D). In contrast, the NF-κB response was attenuated and delayed in TLR2D mice. In wild-type mice, the S aureus–activated NF-κB complex consisted of p65 and p50 NF-κB heterodimers.
In wild-type mice, AP-1 activity steadily increased after S aureus challenge (Figure 6A). In contrast, the degree of activation was reduced in TLR2D mice. JNK activity also increased over time in wild-type mice after S aureus challenge (Figure 6B). Hearts from TLR2D mice, on the other hand, exhibited an attenuated JNK response to S aureus, demonstrating that TLR2 participates in the induction of JNK activity in cardiac tissue.
TLR2 Mediates the LV Dysfunction Induced by S aureus
The increased susceptibility of TLR2D mice to S aureus infection, as well as their increased mortality, precluded assessment of myocardial function in vivo.15 Therefore, the contractile responses of hearts from S aureus–challenged wild-type (n=5) and TLR2D (n=5) mice were assessed ex vivo. There were no differences in baseline contractile function between hearts from wild-type (n=8) and TLR2D (n=8, Table) mice. Wild-type S aureus–treated hearts exhibited significant (P≤0.05) contractile dysfunction compared with buffer-perfused hearts from wild-type mice (Figure 7). In marked contrast, hearts from TLR2D mice were protected against S aureus–induced contractile dysfunction. That is, S aureus–challenged hearts from TLR2D animals showed no significant decreases in maximal developed LV pressure. The preservation of LV function in TLR2D mice was associated with reduced TNF (4.4±1.7 in TLR2D versus 12.1±2.8 pg/mg in wild-type mice, P≤0.05) and IL-1β (2.8±0.506 in TLR2D versus 6.8±1 pg/mg in wild-type mice, P≤0.05) production in Langendorff preparations.
The studies reported here show for the first time that TLR2 signaling contributes to the development of S aureus–induced LV dysfunction in the adult heart. Hearts from wild-type S aureus–challenged mice exhibited reproducible impaired LV function when compared with buffer-perfused hearts. More important, the response of S aureus–challenged hearts from TLR2D mice was not significantly different from that of buffer-perfused hearts from wild-type or TLR2D mice (Figure 7). Given that these studies were performed in isolated, perfused hearts, the results indicate that the LV dysfunction in wild-type mice was due to TLR2-mediated signaling in the heart. Importantly, the observed differences in contractile function in wild-type mice and TLR2D mice were not secondary to baseline differences in LV function (Table). Although the mechanism for the difference in LV function is unknown, the results strongly suggest that blunted expression of proinflammatory mediators may be responsible for the preserved LV function in S aureus–challenged TLR2D mice. We have demonstrated that cardiac NF-κB activation was attenuated and delayed and that TNF and IL-1β levels were significantly lower (in vivo and ex vivo) in S aureus–challenged TLR2D mice.
TNF has been implicated as an endogenous myocardial depressant, and IL-1β can synergistically accentuate this effect.7,8 Thus, protection against LV dysfunction (ex vivo) in TLR2D mice may result from diminished cardiac TNF expression in these mice compared with wild type. Thomas et al21,22 have recently shown that deletion of IRAK1 leads to blunted LPS signaling in the heart and protects against LPS- or burn-induced myocardial dysfunction. Although at the present time it is not possible to completely discern whether the cardioprotection afforded in this model results from defective TLR2 signaling in the heart or other tissues, the data presented strongly suggest that TLR2 signaling can regulate cardiac function in the setting of staphylococcal sepsis.
In mammalian species there are at least 10 TLRs, and each has a distinct function in innate immune recognition.23 Once triggered by microbial antigens, signaling occurs by a pathway that uses components shared among both TLRs and the IL-1 receptor family. It initially had been proposed that the TNF response induced by S aureus and its cell wall components involved the engagement of CD14.24 However, studies in CD14-deficient mice revealed that TNF levels in these mice were 3-fold higher than in wild-type mice after S aureus infection, suggesting that CD14 may downregulate TNF production.25 The requirement for TLR2 in S aureus recognition was later established, as TLR2D mice were less responsive (TNF production) to whole S aureus and peptidoglycan than were wild-type animals.15 The observation in this study that TLR2 deficiency blunted rather than abrogated the inflammatory response suggests that other TLRs or TLR-independent pathways may contribute to S aureus–induced cytokine production in the heart. Although we did not detect increases in cardiac TLR4 expression, this receptor can mediate the inflammatory response to lipoteichoic acid, a component of the S aureus cell wall.14 We also considered that residual responses in TLR2D mice may result from the low levels of IL-1β protein produced, as IL-1 receptor and TLR2 signaling converge at MyD88.
Although the aforementioned discussion focuses attention on the deleterious effects of TLR2 expression in the heart, the observation that S aureus–infected TLR2D mice had a higher mortality than did wild-type mice suggests that expression of TLR2 by immune cells assists in the early recognition and resolution of bacteremia.15 The finding that S aureus infection leads to an acute increase in TLR2 expression in the heart suggests that it may be a key regulator of the compartmentalized production of proinflammatory mediators known to cause myocardial dysfunction. As in the present study, Williams et al26 reported increases in TLR2 gene expression in the liver and lung that were correlated with increased mortality in a mouse model of septic peritonitis. Thus, the time course of TLR2 regulation in cardiac and other tissues needs to be investigated further, as it may determine a window of opportunity for possible therapeutic interventions.
In conclusion, this study has shown for the first time that TLR2 is necessary for the increased expression of inflammatory mediators and the development LV dysfunction during S aureus sepsis. Thus, pharmacological agents that would allow manipulation of common targets within the Toll/IL-1 receptor signaling cascades may represent a novel strategy to protect the heart against the dysfunctional inflammatory response associated with S aureus sepsis.
This research was supported by grants GM62474 from the National Institute of General Medical Sciences and KN521/1-1 from the Deutsche Forschungsgemeinschaft (to P.K.). We thank Douglas L. Mann, MD, and Carol J. Baker, MD, for reviewing the manuscript.
↵*Drs Knuefermann and Sakata contributed equally to this work.
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