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IRAK1 deletion disrupts cardiac Toll/IL-1 signaling and protects against contractile dysfunction

Departments of ¹Pediatrics, ²Molecular Biology, and ³Surgery, The University of Texas Southwestern Medical Center, Dallas, Texas 75390; and ⁴Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210

Submitted 26 July 2001 ; accepted in final form 21 April 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Myocardial contractile dysfunction accompanies both systemic and cardiac insults. Septic shock and burn trauma can lead to reversible contractile deficits, whereas ischemia and direct inflammation of the heart can precipitate transient or permanent impairments in contractility. Many of the insults that trigger contractile dysfunction also activate the innate immune system. Activation of the innate immune response to infection is coordinated by the conserved Toll/interleukin-1 (IL-1) signal transduction pathway. Interestingly, components of this pathway are also expressed in normal and failing hearts, although their function is unknown. The hypotheses that Toll/IL-1 signaling occurs in the heart and that intact pathway function is required for contractile dysfunction after different insults were tested. Results from these experiments demonstrate that lipopolysaccharides (LPS) activate Toll/IL-1 signaling and IL-1 receptor-associated kinase-1 (IRAK1), a critical pathway intermediate in the heart, indicating that the function of this pathway is not limited to immune system tissues. Moreover, hearts lacking IRAK1 exhibit impaired LPS-triggered downstream signal transduction. Hearts from IRAK1-deficient mice also resist acute LPS-induced contractile dysfunction. Finally, IRAK1 inactivation enhances survival of transgenic mice that develop severe myocarditis and lethal heart failure. Thus the Toll/IL-1 pathway is active in myocardial tissue and interference with pathway function, through IRAK1 inactivation, may represent a novel strategy to protect against cardiac contractile dysfunction.

heart failure; signal transduction; contractile function; genetically altered mice

Three types of observations have linked myocardial dysfunction with inflammation. First, acute infections and inflammatory diseases can precipitate transient heart failure (29–32, 39). Second, both microbial and endogenous proinflammatory mediators, such as bacterial endotoxin [lipopolysaccharide (LPS)], interleukin-1 (IL-1), and tumor necrosis factor (TNF)- directly depress myocardial contractility (5, 9, 21, 35, 37, 38). Third, elevated circulating concentrations of these molecules also occur in patients with heart failure of any cause and are correlated with more severe myocardial dysfunction (18, 27, 46).

Expression of the Toll-like receptor-4 (TLR4), the mammalian LPS sensor, together with its intracellular signaling apparatus (6, 7, 10, 13, 23, 33) in the heart, suggests a mechanism whereby LPS could trigger cardiac dysfunction. In immune cells such as macrophages, LPS signals through an intracellular pathway shared with the type I IL-1 receptor (the Toll/IL-1 pathway). After activation of TLR4 or the type 1 IL-1 receptor, MyD88 and IL-1 receptor associated kinase-1 (IRAK1) are recruited to the activated receptor complex (6, 24, 48). IRAK1 becomes phosphorylated, dissociates from the receptor complex, and associates with TNF receptor-associated factor 6 (6, 7). The signal is then distributed to multiple downstream targets, including NF-B, c-Jun NH₂-terminal kinase (JNK), and p38 MAPK. Deletion of MyD88, IRAK1, or TNF receptor-associated factor 6 disrupts both IL-1 and LPS signaling to distal pathways and disturbs normal physiological responses to these proinflammatory mediators in innate immune cells (1, 15, 16, 20, 25, 41, 44).

The presence of Toll/IL-1 signaling molecules in the heart and their upregulation in disease states such as congestive heart failure led to the hypothesis that the same pathway that initiates the inflammatory response to infection in immune cells also functions in the heart and mediates acute and chronic myocardial contractile dysfunction. Although previous studies (26, 45) have demonstrated a role for TLR4 in both LPS- and burn-induced myocardial dysfunction, a requirement for IRAK1 has only been shown in burn-triggered contractile impairment (43). Thus it is unknown whether TLR4 stimulation activates IRAK1 in the heart or whether IRAK1 function is necessary for contractile dysfunction after LPS administration. Studies reported here show that LPS activates IRAK1 in the heart. Moreover, IRAK1 inactivation alters cardiac signaling responses to LPS and protects mice from LPS-induced contractile function. Finally, IRAK1 deletion enhances survival in transgenic mice with myocarditis, cardiomyopathy, and lethal heart failure. Thus interference with Toll/IL-1 signaling, as occurs with IRAK1 inactivation, may represent a therapeutic strategy to ameliorate conditions associated with either acute or chronic cardiac contractile dysfunction.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Reagents. Escherichia coli LPS (O111:B4, Sigma) was used for in vivo injections. Antisera against IRAK1 and JNK were provided by Zhaodan Cao (Tularik) and Melanie Cobb (University of Texas Southwestern), respectively. Polyclonal antisera against IKK- and IB- were purchased from Santa Cruz Biotechnology and New England Biolabs, respectively. Recombinant glutathione S-transferase-IB- and glutathione S-transferase-c-Jun were provided by Melanie Cobb.

Animals. All animals were used in compliance with the guidelines established by the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center and performed in accordance with National Institutes of Health guidelines for the use of laboratory animals.

IRAK1-deficient and cardiac-specific TNF- overexpressor mice were generated and genotyped as previously described (4, 44). For generation of the IRAK1 knockout (KO), the Irak1 gene was inactivated in murine embryonic stem cells using targeted mutagenesis. Mutant stem cells were injected into wild-type (WT) blastocysts and resulted in chimeric mice. Two chimeric animals transmitted the mutant Irak1 gene through the germline. Offspring from these founders were interbred to generate homozygous IRAK1-deficient females and hemizygous IRAK1-deficient males (Irak1 is located on the X chromosome) (44). These animals were on a hybrid background (129Sv x C57BL/6). WT animals on a similar hybrid background were used as controls for all experiments. Cardiac-specific TNF--overexpressing mice were generated by microinjection of a transgene encoding the murine TNF- sequence coupled to an -myosin heavy chain promoter into the male pronucleus of fertilized mouse eggs and implanted into pseudopregnant females. Offspring with the integrated transgene became founders for this novel line of transgenic mice. To generate transgenic KO double mutant mice, homozygous IRAK1-deficient females were crossed to male TNF- overexpressors. Male transgenic offspring were crossed to IRAK1 KO females and progeny with the TNF- transgene were used in subsequent studies. Hybrid (C57Bl/6J x SJL) WT mice also served as additional controls.

In vitro kinase reactions. At different times after LPS injection, mice were euthanized, and the hearts were immediately removed and snap frozen in liquid nitrogen. Longitudinal sections (25–40 mg) that included both atrial and ventricular tissue were cut from frozen hearts, rinsed in ice-cold phosphate-buffered saline to remove clotted blood, and minced into small pieces using a number 11 scalpel blade. The tissue was then disrupted in a Dounce homogenizer with 1 ml of either a HEPES lysis buffer [50 mM HEPES, pH 7.5; 150 mM NaCl; 1% Triton X-100; 10% glycerol, and 1 mM dithiothreitol (DTT)] with protease (Complete Inhibitor, Roche; Indianapolis, IN) and phosphatase inhibitors (20 mM NaF, 20 mM sodium glycerophosphate, and 0.5 mM sodium orthovanadate) for all kinases except IRAK1, or a RIPA lysis buffer (50 mM Tris · HCl, pH 7.4; 150 mM NaCl; 1% Nonidet P-40; 0.25% sodium deoxycholate; 0.1% SDS; and 1 mM DTT) with the same protease and phosphatase inhibitors. After a 30-min incubation on ice, lysates were cleared by centrifugation at 20,000 g for 10 min at 4°C, and the supernatant was transferred to a clean microcentrifuge tube. The total protein concentration of the lysates was determined with the use of a protein assay kit (Bio-Rad; Hercules, CA). Kinases were immunoprecipitated from 1 mg of total cardiac protein in a minimum 250-µl volume with 1 to 5 µl of antiserum on rocking platform overnight at 4°C. Protein A-conjugated agarose (20 µl; Roche) was added and allowed to incubate for 2 h at 4°C. Precipitates were washed three times with lysis buffer and once with incomplete kinase buffer composed of (in mM) 50 Tris · HCl (pH 7.4), 10 MgCl₂, and 1 DTT. Beads were then resuspended in complete kinase buffer composed of 50 mM Tris · HCl (pH 7.4), 10 mM MgCl₂, 1 mM DTT, 50 µM "cold" ATP, and [-³²P]ATP (10 µCi/reaction) together with appropriate substrate (0.3 mg/ml) in a total volume of 25 µl. The mixtures were incubated at 30°C for 30 min. Reactions were stopped by adding an equal volume of 2x SDS-PAGE sample buffer (200 mM Tris · HCl, 4% SDS, 0.04% bromophenol blue, and 20% glycerol), followed by heating at 95°C for 5 min. Samples were fractionated on a large-format 10% SDS-polyacrylamide gel and the proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore; Bedford, MA). Membranes were air dried, exposed to phosphor storage screens, and developed with the use of a phosphorimaging system (Molecular Dynamics; Mountain View, CA). Fold catalytic activation was determined by densitometric increases in signal above the 0-h time point.

Electromobility shift assays. A modified procedure based on the method of Schreiber et al. (34) was used. Longitudinal heart sections (25 mg) were homogenized in ice-cold hypotonic lysis buffer composed of (in mM) 10 Tris · HCl (pH 7.8), 5 MgCl₂, 10 KCl, 0.3 EGTA, and 0.5 DTT and 0.3 M sucrose (protease inhibitors) and incubated on ice for 15 min. After the addition of Nonidet P-40 (final concentration: 0.5%) the mixture was vortexed and centrifuged to recover nuclei. The supernatant was saved for IB immunoblots. Nuclear proteins were extracted from the pellet that was composed of (in mM) 20 Tris · HCl (pH 7.8), 5 MgCl₂, 320 KCl, 0.2 EGTA, and 0.5 DTT (plus protease inhibitors) for 15 min on ice and centrifuged at 13,500 g for 15 min. Extracts were stored at –80°C. Total protein concentration of nuclear extracts was determined with a protein assay kit (Bio-Rad).

Double-stranded oligonucleotide corresponding to the consensus NF-B binding site of the murine -light chain enhancer (5'-AGTTGAGGGGACTTTCCCAGGC-3') was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Oligonucleotide (3.5 pmol), T4 polynucleotide kinase (5 units) in 1x kinase buffer (Promega; Madison, WI), and [-³²P]ATP (30 µCi; DuPont-New England Nuclear; Boston, MA) were incubated at 37°C for 60 min. The labeled probe was separated from unbound ATP with the use of microcolumns (ProbeQuant G-50, Amersham Pharmacia Biotech) and stored at –20°C. Nuclear proteins (2.5 µg) were incubated with 500,000 counts/min of probe in the presence of salmon sperm DNA (2 µg) in 1x gel shift buffer composed of (in mM) 20 HEPES, 50 KCl, 1 DTT, and 1 EDTA (pH 7.6) and 5% glycerol for 30 min at room temperature. The mixtures were then separated on a nondenaturing 8% polyacrylamide gel in 0.5x 25 mM Tris · HCl, 25 mM boric acid, and 0.5 mM EDTA. The gel was dried and exposed to X-ray film (Kodak BioMax).

Immunoblots. Samples containing cytosolic protein (50 µg) were fractionated on a 12% SDS-polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore). Membranes were washed in 1x Tris-buffered saline (TBS; 20 mM Tris · HCl and 140 mM NaCl, pH 7.5), blocked for 1 h at room temperature (1x TBS, 0.1% Tween 20, and 5% nonfat dry milk), and then incubated with primary antibody (rabbit polyclonal IB antibody, Santa Cruz Biotechnology) 1:1,000 in dilution buffer (1x TBS, 0.1% Tween 20, and 5% nonfat dry milk) overnight at 4°C. Membranes were washed four times in 1x TBS and 0.1% Tween 20, followed by incubation with peroxidase-labeled anti-rabbit IgG (1:8,000 in dilution buffer, Santa Cruz) for1hat room temperature. Membranes were washed four times at room temperature before the antigen-antibody complexes were detected with enhanced chemiluminescence (Super Signal, Pierce; Rockford, IL).

Langendorff-perfused hearts. To examine cardiac contractile function, mice were divided into four experimental groups. Group 1 consisted of sham-treated WT mice (n = 5). Group 2 was composed of sham-treated IRAK1 KO animals (n = 7). Group 3 was LPS-treated WT mice (n = 9), and group 4 was LPS-treated IRAK1 KO animals (n = 10). Separate groups of alert mice from all experimental groups were anti-coagulated with heparin sodium (100 units, Elkins-Sinn, Cherry Hill, NJ) and euthanized by cervical dislocation. The heart was rapidly removed and placed in ice-cold (4°C) Krebs-Henseleit bicarbonate-buffered solution composed of (in mM) 118 NaCl, 4.7 KCl, 21 NaHCO₃, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, and 11 glucose. All solutions were prepared each day with demineralized, deionized water and bubbled with 95% O₂-5% CO₂ (pH 7.4; PO₂, 550 mmHg; PCO₂, 38 mmHg). Polyethylene-50 (PE-50) tubing (Intramedic) was placed in the ascending aorta and connected via glass tubing to a buffer-filled reservoir for perfusion of the coronary circulation at a constant flow rate. Hearts were suspended in a temperature-controlled chamber maintained at 38.6 ± 0.5°C, and the coronary arteries were perfused by retrograde flow through the aortic stump cannula with the use of a constant flow pump (model TIA, Ismatec, Cole Palmer; Vernon Hills, IL). Contractile function was assessed by measuring intraventricular pressure with the PE-50 tubing threaded into the left ventricle (LV). LV pressure (LVP) was measured with a Statham pressure transducer (model P23 ID, Gould Instruments; Oxnard, CA) attached to the cannula, and the rates of LVP rise (+dP/dt) and fall (–dP/dt) were obtained using an electronic differentiator (model 7P20C, Grass Instruments, Quincy, MA), recorded (model 7DWL8P, Grass Recording Instruments), and transferred to a Dell Pentium computer. Contractile function was determined by plotting LV systolic pressure and ±dP/dt_max values against increases in coronary flow, achieved by increasing the flow rate of a pump calibrated to deliver between 1 and 4 ml/min into the cannulated aortic stump, or perfusate Ca²⁺ concentration. After removal, cardioplegia, cannulation, and perfusion, ex vivo denervated hearts typically beat between 280 and 310 beats/min. If the rhythm was too slow, too fast, or irregular, the hearts were captured at their intrinsic rates and then paced at 300 beats/min through an electrode attached to the right atrium (4.8–5.0 A for 1-ms duration, Grass Stimulator) for the duration of the contractile function studies. There is no difference between groups with regard to the requirements for pacing.

Histology. Hearts were fixed by immersion in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 6 µm. Sections were stained with hematoxylin and eosin for routine examination and with Masson's trichrome to reveal collagen. Coded samples were scored by a single veterinary pathologist (D. F. Kusewitt) located at another institution for severity of myocarditis on a scale of 0–3 (0, no myocarditis; 1, mild myocarditis; 2, moderate myocarditis; 3, severe myocarditis). The reader was blinded to the animals' genotypes. Severity scores were based on the extent of myocardial involvement and the degree of interstitial cellularity and fibrosis in affected areas. Less than 25% involvement of the heart was scored as mild myocarditis; in cases of moderate myocarditis, 25–50% of the heart was affected; in cases of severe myocarditis, >50% of the heart was involved.

Statistics. Separate analyses of cardiac functions were conducted for each of LVP, +dP/dt, and –dP/dt as a function of treatment group (factor 1: WT sham vs. LPS, KO sham vs. LPS, sham WT vs. KO) and either coronary flow rate or calcium level (factor 2). A two-way analysis of variance was performed. In those instances in which the factor 1–2 interaction was significant at the 0.05 level, Bonferroni-corrected post tests were then conducted at each level of factor 2 (coronary flow rate or perfusate calcium) to discern differences among the treatment groups. Survival curves of TNF- transgenic and transgenic/IRAK1-deficient mice were compared using a Kaplan-Meier survival estimate.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
IRAK1 is activated in heart by LPS and is required for optimal downstream signaling. To determine whether LPS activates IRAK1 in the heart, WT mice were challenged with LPS and IRAK1 catalytic activity was measured using an immunoprecipitation in vitro kinase assay. Thirty minutes after LPS treatment, IRAK1 catalytic activity increased about threefold above the unstimulated state (Fig. 1A) and declined thereafter (data not shown). The contribution of IRAK1 to activation of two downstream signaling pathways in the heart was then examined. LPS can cause rapid cardiac NF-B activation (14). The responses of hearts from WT and IRAK1-deficient mice (44) were then compared. Animals were injected with LPS (1 mg/kg ip), their hearts were removed, and NF-B activation was examined at various times. Catalytic activation of IKK-, the kinase that phosphorylates IB- and targets it for degradation, was assayed first. As shown in Fig. 1B, IKK- catalytic activity increased rapidly in WT hearts, reached 15-fold activation after 30 min, and then declined, returning to baseline by 3 h. In contrast, IKK- activity in IRAK1-deficient hearts was diminished and delayed compared with WT, with peak activation sevenfold above baseline. Similar results were obtained in two other trials. Thus IRAK1 governs both the kinetics and peak activity of the principle kinases controlling NF-B activation in the heart, an effect similar to that seen in LPS-treated macrophages (41). This finding is also reflected by the delayed and incomplete LPS-induced IB- degradation (Fig. 1C) and reduced NF-B DNA binding activity in IRAK1-deficient compared with WT hearts (Fig. 1D). Together, these findings demonstrate that LPS-mediated NF-B activation in the heart operates through an IRAK1-dependent pathway.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Lipopolysaccharide (LPS) activation of intracardiac signaling pathways. A: LPS induction of interleukin-1 receptor-associated kinase-1 (IRAK1) catalytic activity in the heart. IRAK1 was immunoprecipitated (IP) from cardiac tissue of wild-type (WT) saline- or LPS (1 mg/kg ip)-treated mice and incubated in the presence of [-32P]ATP and myelin basic protein (MBP). B: impaired IKK- activation in IRAK1–/– (knockout; KO) hearts. Hearts from LPS-treated WT and KO mice were assayed for IKK- kinase activity using an IP kinase assay with recombinant IB- as the substrate. C: delayed and incomplete IB- degradation in IRAK1-deficient hearts. Equal amounts of protein from WT and KO heart cytoplasmic extracts were immunoblotted with a commercially available anti-serum against IB-. WB, Western blot. D: optimal nuclear factor-B (NF-B) activation requires IRAK1. Nuclear extracts were incubated with a radioactively labeled oligonucleotide containing an NF-B consensus binding sequence and fractionated on a native polyacrylamide gel. E: requirement for IRAK1 in LPS induction of cardiac c-Jun NH2-terminal kinase (JNK) activation. Hearts from LPS-treated WT and KO mice assayed for JNK catalytic activity with an IP kinase assay using recombinant c-Jun as substrate. GST, glutathione S-transferase. Extracts used for assays in B–E come from the same hearts. Data shown are from one of three representative experiments.

The contribution of IRAK1 to JNK signaling in the heart was also examined. JNK activity controls LPS-induced TNF- production in macrophages (12, 40). This cytokine is produced in cardiac myocytes after LPS stimulation and causes myocardial contractile dysfunction (4, 5, 38). LPS treatment of WT mice triggered a 28-fold increase in cardiac JNK activity in the experiment shown in Fig. 1E. Peak activation that occurred 15 min after injection returned to basal levels by 60 min. IRAK1-deficient hearts, on the other hand, exhibited a severely attenuated and delayed response to LPS injection (Fig. 1E), demonstrating that IRAK1 is required for LPS-induced JNK activation in heart tissue.

IRAK1 mediates LPS-induced myocardial contractile dysfunction. Sepsis causes myocardial contractile dysfunction (30, 32), and administration of endotoxin to both humans and experimental animals mimics the myocardial depression of sepsis (30, 39). To determine whether differences in intracellular signaling were also associated with altered myocardial contractile function, the contractile responses of hearts from LPS-treated WT and IRAK1-deficient mice were compared. Animals were injected with either LPS (1 mg/kg ip) or an equal volume of normal saline. Eighteen hours later, the hearts were removed and contractile function was assessed ex vivo with the use of modified Langendorff-isolated perfusion preparations. There was no difference in baseline contractile function between hearts from WT or IRAK1-deficient mice. Both exhibited similar systolic maximal developed LVP and rate of pressure generation (+dP/dt_max) and diastolic rate of relaxation (–dP/dt_max) responses to increasing coronary flow rates (Fig. 2) and perfusate calcium concentrations (Fig. 3A). As expected, WT LPS-treated hearts exhibited significant myocardial contractile dysfunction compared with sham-treated WT hearts. This impaired function affects both systole and diastole and persists at increasing coronary flow rates (Fig. 2) and extracellular calcium concentrations (Fig. 3). In marked contrast, IRAK1-deficient hearts are protected against LPS-induced contractile dysfunction. Hearts from LPS-treated IRAK1-deficient animals showed no significant decreases in maximal developed LVP, +dP/dt_max, or –dP/dt_max in response to increasing coronary flow rates (Fig. 2). Similarly, hearts from IRAK1-deficient mice challenged with LPS were also resistant to the myocardial depressant effects of LPS, as determined by their systolic and diastolic responses to increasing calcium concentrations (Fig. 3). Thus IRAK1 inactivation blocks acute LPS-triggered cardiac contractile failure, implicating this kinase as a transducer of the acute myocardial depressant effects of endotoxin.

View larger version (30K): [in this window] [in a new window]   Fig. 2. IRAK1 mediates LPS-induced myocardial contractile responses to increasing coronary flow rates. Systolic and diastolic contractile function of hearts from saline- and LPS-treated (1 mg/kg ip) WT and IRAK1-deficient mice were determined using a modified Langendorff-isolated heart perfusion preparations. Maximal developed left ventricular pressure (LVP; A), rate of pressure generation (+dP/dtmax; B), and rate of relaxation (–dP/dtmax; C) responses of hearts to increased coronary flow were determined as described. The responses of untreated WT (n = 5) and KO (n = 7) mice, as well as saline- and LPS-treated WT (n = 9) or KO (n = 10) mice, were compared using ANOVA, followed by corrected t-tests after detection of a significant interaction between different factors (see MATERIALS AND METHODS). Data shown represent means ± SE. *P < 0.001, P < 0.05.

View larger version (24K): [in this window] [in a new window]   Fig. 3. A: IRAK1 mediates LPS-induced myocardial contractile responses to increasing extracellular Ca2+ concentrations. B and C: responses of isolated perfused WT and KO hearts from saline- and LPS-treated animals to increasing perfusate Ca2+ concentrations were compared as described in Fig. 2. Data shown represent means ± SE. *P < 0.001, P < 0.01.

IRAK1 inactivation prolongs survival in mice that overexpress TNF- in the heart. Although IRAK1 is crucial to acute LPS-induced myocardial depression, its role in conditions characterized by chronically impaired contractility is unknown. To determine whether IRAK1 contributes to chronic contractile dysfunction as well, IRAK1 was inactivated in transgenic mice with a lethal form of heart failure. IRAK1-deficient mice were crossed with transgenic mice that overexpress TNF- in the heart. These TNF- transgenic animals develop myocarditis, dilated cardiomyopathy, heart failure, and experience 100% early lethality (4). Survival of the transgenic animals and the TNF- transgenic/IRAK1 KO double mutants was compared. Figure 4A depicts the survival curves of the two strains of animals. Elimination of IRAK1 function markedly enhanced the 150-day survival of TNF- transgenic mice. All of the transgenic animals with functional IRAK1 succumbed during the test period, whereas 70% of those lacking the kinase were still alive after 150 days. Therefore, IRAK1 inactivation protects mice against this form of lethal heart failure.

View larger version (88K): [in this window] [in a new window]   Fig. 4. IRAK1 deletion prevents death in lethal congestive heart failure. A: IRAK1 KO mice were interbred with animals overexpressing a tumor necrosis factor- (TNF-) transgene (TG) exclusively in the heart, and survival and time to death were compared using a Kaplan-Meier survival estimate. The experiment was terminated when the animals reached 150 days of age. B: histological appearance of hearts. Hearts of WT, IRAK1 KO, TNF- TG, TNF- TG/IRAK1 KO double mutant mice underwent blinded histological evaluation. Interstitial hypercellularity and fibrosis characterized moderate to severe myocarditis in the hearts of TNF- transgenic mice and double mutants. Although myocarditis was generally diffuse, the foci of hypercellularity (arrows) were also present. Inset: Anitschkow-like cells in the heart of a TNF- TG mouse.

Because heart failure in the transgenic mice results from cardiac overexpression of the proinflammatory cytokine TNF-, and IRAK1 also mediates injury sensing and inflammation at several different steps in the innate immune response, it is possible that decreased mortality was due to a diminished inflammatory response in transgenic/KO hearts. On average, hearts from 75-day-old WT mice and IRAK1 KO mice appeared normal in size and consistency. In contrast, hearts from the TNF- overexpressors were markedly dilated, thin walled, and pale. Most hearts from double mutants (TNF- transgenic/IRAK1 KO) appeared normal, but occasionally (<10%) a transgenic/KO animal would have a dilated heart similar to the transgenic animals. These rare animals were also clinically ill. Hearts from WT (n = 4), TNF- transgenic (n = 7), IRAK1 KO (n = 7), and TNF- transgenic/KO (n = 4) mice were removed for histopathological examination by a veterinary pathologist who was blinded to the animals' genotypes. Hearts were classified as having no, mild, moderate, or severe myocarditis (Table 1). WT and IRAK1-deficient hearts lacked evident cardiac lesions (Fig. 4B). Hearts from TNF- transgenic mice exhibited diffuse interstitial cellularity, with mononuclear inflammatory cells and fibroblasts, as well as interstitial fibrosis in both atria and ventricles, as previously described (4). Cardiac myocytes from these transgenic animals showed degenerative changes, including cytoplasmic vacuolization and hyalinization, nuclear enlargement, vesiculation, and the development of Anitschkow-like nuclear changes (Fig. 4B). TNF- transgenic/IRAK1 KO hearts were histologically indistinguishable from TNF- transgenic hearts and exhibited either moderate or severe myocardial lesions (Fig. 4B). Thus, on the basis of histological evaluation, loss of IRAK1 function does not attenuate cardiac inflammation in mice overexpressing TNF- in cardiomyocytes.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The studies reported here show for the first time that LPS activates IRAK1 in the heart. Moreover, hearts lacking IRAK1 exhibit impaired activation of the pathways that result in NF-B and JNK activity, displaying diminished amplitude and timing of downstream signaling required for TNF- production in other cells (40). These findings are consistent with previously published reports. Giroir and colleagues (11) first demonstrated LPS-driven cardiac TNF-, whereas Frantz and colleagues (10) have shown TLR4 expression in normal myocytes and failing hearts, and Baumgarten et al. (3) have implicated TLR4 in this cardiac-specific TNF- response. Activation of cardiac NF-B by LPS has also been reported previously (3, 8, 14, 36) with the observation that NF-B inhibition protects against LPS-induced myocardial contractile dysfunction (8, 14, 36). The present study demonstrates a requirement for the Toll/IL-1 signaling intermediate IRAK1 in the cardiac signaling response to LPS. Moreover, LPS-induced JNK catalytic activation in the heart and the effect of IRAK1 inactivation on cardiac JNK signaling also represents a novel finding. In fact, WT and IRAK1-deficient hearts exhibit endotoxin-triggered signaling profiles that resemble those seen in macrophages (41), which also showed attenuated and delayed NF-B and MAPK responses. The macrophage studies, however, were conducted in vitro, so there may be important differences in signal transduction between these two tissues. Thus the myocardium possesses an LPS-sensing apparatus, and IRAK1 regulates the cardiac signaling response to endotoxin.

The results from these studies also implicate IRAK1 in acute LPS-induced contractile dysfunction. WT LPS-treated hearts exhibited predictable and reproducible impaired contractility compared with sham-treated hearts, whereas the response of LPS-challenged IRAK1-deficient hearts was no different from sham-treated hearts lacking IRAK1. Thus IRAK1 function is required for the development of LPS-induced contractile depression.

These findings reinforce the importance of Toll/IL-1 signaling in acute myocardial contractile dysfunction by demonstrating its requirement in a second model of cardiac depression. Recent work in our laboratory (43) has demonstrated that IRAK1 transduces a signal that leads to myocardial dysfunction after large body surface area burns (35–40% total body surface area). Additional studies indicated that TLR4 was a principal sensor for burn-induced myocardial dysfunction because mice harboring inactivating TLR4 mutations failed to develop cardiac depression after injury (45). Furthermore, contractile dysfunction after burn injury could be prevented by administrations of the LPS inhibitor recombinant bactericidal/permeability-increasing protein (rBPI₂₁) (45). Together, the results from the burn studies suggested that myocardial depression might be due to LPS translocated from the gut of injured animals, although other possible mechanisms could not be excluded. Regardless of whether LPS is responsible for myocardial depression in burn injury, the findings presented in the current experiments document a clear role for IRAK1 in contractile dysfunction after acute exogenous endotoxin administration.

In addition to its effect on acute LPS-mediated contractile dysfunction, IRAK1 may also be involved in chronically impaired contractility. IRAK1 inactivation enhances survival in animals with congestive heart failure due to cardiac-specific TNF- overexpression. How IRAK1 deletion exerts this protection is unknown. Histological analysis suggests that IRAK1 deletion does not directly affect the degree of inflammation seen in transgenic hearts. Double mutant animals still synthesize TNF- in the heart and still exhibit an inflammatory infiltrate similar to transgenic mice with IRAK1, but nonetheless live longer. Interestingly, contractile function in transgenic animals at 50 days of life, the time when some of these animals begin to die is significantly impaired compared with double mutant animals of the same age (data not shown), but this additional observation fails to reveal a mechanism linking IRAK1 activation to worsening heart failure.

Two general possibilities could help explain this result. First, IRAK1 may be required for normal TNF- signaling. Most studies to date have failed to uncover a role for IRAK1 in TNF- signaling, although one report has suggested that IRAK1 potentiates IL-1 signaling to NF-B (47). According to this view, loss of IRAK1 function would attenuate cardiac responses to TNF-, thereby lessening the degree of contractile dysfunction in transgenic hearts lacking IRAK1. Alternatively, IRAK1 may promote lethality by transducing other, TNF--independent signals in progressive heart failure. Importantly, patients with severe (New York Hospital Association class III and IV) congestive heart failure and edema exhibit elevated serum LPS and proinflammatory cytokine concentrations (18, 27), which may be both markers for worsening cardiac function and stimuli contributing to further deterioration in contractility. Normally, the presence of endotoxin would be expected to worsen contractile function, if the host is insensitive to this compound; however, as occurs in the double mutant mice, myocardial function might be spared. Therefore, a loss of IRAK1 function through genetic deletion may abrogate three major proximal signals that trigger cardiac dysfunction. Direct myocardial depressant actions of LPS and IL-1 are blocked, and LPS-induced TNF- production by noncardiac cells is also impaired, such that the heart is exposed to less circulating TNF-. Loss or diminution of these three negative inotropic inputs may slow the deterioration of contractile function in double mutant mice and thereby prolong survival. Determining the precise role for IRAK1 in lethality is this model of heart failure; however, it will require further study.

Although they identify IRAK1 as an important participant in LPS-induced myocardial dysfunction, these studies cannot pinpoint the site of IRAK1 action in this response. Myocardial protection is correlated with altered myocardial signaling responses to LPS, but it is impossible to discern whether this protection stems from disrupted signaling in the heart or some other tissue(s). LPS responsiveness is presumably disrupted in all IRAK1-deficient tissues. Thus these studies cannot determine whether the myocardial protection seen in KO mice is due to the lack of IRAK1 in the heart of its absence from some other cell type. How, then, does LPS cause myocyte contractile dysfunction? Two models, both testable, can be envisioned. In the first, LPS sensing and impaired contractility both occur in the cardiac myocyte (a cell-autonomous model). Conversely, nonmyocyte cell types either in the heart itself or elsewhere (e.g., fibroblasts, macrophages, monocytes, endocardial or endothelial cells, or hepatocytes) could sense LPS and produce secondary signals (e.g., cytokines such as TNF- or IL-1 or other molecules) that directly depress myocardial contractility (a cell nonautonomous model). Furthermore, the models are not mutually exclusive.

Regardless of the site of IRAK1 action, the data presented here suggest that a signaling pathway considered part of the innate immune system can regulate a nonimmune physiological response. IRAK1 inactivation blocks the usual negative effect of LPS, a bacterial product with well-described immunostimulatory properties, on cardiac contractility. Most studies of Toll/IL-1 signaling have focused on the immune consequences of pathway stimulation. This report, however, represents one of the earliest attempts to link pathway activation to physiological alterations outside the immune system. These findings have two important implications. First, demonstration that IRAK1 regulates LPS-induced contractile depression highlights the interrelatedness of different systems in integrated biological responses to infection. Second, it may force reconsideration of what properly constitutes innate immunity, usually viewed as a form of host defense mediated by bone marrow-derived cells and specialized molecules to aid in antibacterial responses, thrombosis, and inflammation.

Why, for example, does the heart, rarely a direct target of infection, have a functional Toll/IL-1 pathway? Could Toll/IL-1 signaling enact a broader injury-sensing role not limited to the detection of infection, with physiological consequences extending beyond the immune system? Recent reports seem to support this possibility. Several groups have implicated endogenous molecules, such as highly conserved heat shock proteins (28), the breakdown products of the extracellular matrix (42), and products released from necrotic cells (19) as activators of Toll/IL-1 signaling. It is therefore possible that certain Toll-like receptors on neighboring viable heart cells actually sense direct cardiac injury resulting from ischemia or trauma, when necrotic myocytes release their contents? Subsequent pathway activation could trigger responses in uninjured cells that might protect them from injury, lead to the characteristic postinjury inflammatory infiltrate, or precipitate postinjury dysfunction, to name a few possible responses. Thus the Toll/IL-1 pathway may represent a ubiquitous, generic injury sensor that serves to alert the host to damage to one of its parts and initiate the containment and healing responses.

Finally, these studies raise the possibility that IRAK1 or other members of the Toll/IL-1 signaling pathway may be targets for the therapy of cardiac contractile dysfunction. Pinpointing how IRAK1 exacerbates contractile dysfunction and limits survival represent critical first steps in developing such a strategy. The protection that genetic inactivation of IRAK1 affords mice with either acute or chronic contractile dysfunction, however, provides a theoretical basis for the development of new therapies for congestive heart failure. It may someday be possible, for example, to prolong survival in patients with end-stage heart failure through pharmacological IRAK1 inhibition.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This work was supported in part by National Institutes of Health grants (to J. W. Horton, J. A. Thomas, and B. P. Giroir).

	ACKNOWLEDGMENTS

 
The authors thank several colleagues for thoughtful manuscript review.

Present address of S. B. Haudek: Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. A. Thomas, Depts. of Pediatrics and Molecular Biology, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9063 (E-mail: James.Thomas{at}UTSouthwestern.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

1. Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sakagami M, Nakanishi K, and Akira S. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9: 143–150, 1998.[ISI][Medline]
2. Aikawa N, Martyn JA, and Burke JF. Pulmonary artery catheterization and thermodilution cardiac output determination in the management of critically burned patients. Am J Surg 135: 811–817, 1978.[ISI][Medline]
3. Baumgarten G, Knuefermann P, Nozaki N, Sivasubramanian N, Mann DL, and Vallejo JG. In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4. J Infect Dis 183: 1617–1624, 2001.[ISI][Medline]
4. Bryant D, Becker L, Richardson J, Shelton J, Franco F, Peshock R, Thompson M, and Giroir B. Cardiac failure in transgenic mice with myocardial expression of tumor necrosis factor-alpha. Circulation 97: 1375–1381, 1998.[Abstract/Free Full Text]
5. Cain BS, Meldrum DR, Dinarello CA, Meng X, Joo KS, Banerjee A, and Harken AH. Tumor necrosis factor-alpha and interleukin-1beta synergistically depress human myocardial function. Crit Care Med 27: 1309–1318, 1999.[ISI][Medline]
6. Cao Z, Henzel WJ, and Gao X. IRAK: a kinase associated with the interleukin-1 receptor. Science 271: 1128–1131, 1996.[Abstract]
7. Cao Z, Xiong J, Takeuchi M, Kurama T, and Goeddel DV. TRAF6 is a signal transducer for interleukin-1. Nature 383: 443–446, 1996.[Medline]
8. Dawn B, Xuan YT, Marian M, Flaherty MP, Murphree SS, Smith TL, Bolli R, and Jones WK. Cardiac-specific abrogation of NF-kappa B activation in mice by transdominant expression of a mutant I kappa B alpha. J Mol Cell Cardiol 33: 161–173, 2001.[ISI][Medline]
9. Flesch M, Kilter H, Cremers B, Laufs U, Sudkamp M, Ortmann M, Muller FU, and Bohm M. Effects of endotoxin on human myocardial contractility involvement of nitric oxide and peroxynitrite. J Am Coll Cardiol 33: 1062–1070, 1999.[Abstract/Free Full Text]
10. Frantz S, Kobzik L, Kim YD, Fukazawa R, Medzhitov R, Lee RT, and Kelly RA. Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium. J Clin Invest 104: 271–280, 1999.[ISI][Medline]
11. Giroir BP, Johnson JH, Brown T, Allen GL, and Beutler B. The tissue distribution of tumor necrosis factor biosynthesis during endotoxemia. J Clin Invest 90: 693–698, 1992.[ISI][Medline]
12. Hambleton J, Weinstein SL, Lem L, and DeFranco AL. Activation of c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated macrophages. Proc Natl Acad Sci USA 93: 2774–2778, 1996.[Abstract/Free Full Text]
13. Hardiman G, Rock FL, Balasubramanian S, Kastelein RA, and Bazan JF. Molecular characterization and modular analysis of human MyD88. Oncogene 13: 2467–2475, 1996.[ISI][Medline]
14. Haudek SB, Spencer E, Bryant DD, White DJ, Maass D, Horton JW, Chen ZJ, and Giroir BP. Overexpression of cardiac IB prevents endotoxin-induced myocardial dysfunction. Am J Physiol Heart Circ Physiol 280: H962–H968, 2001.[Abstract/Free Full Text]
15. Kanakaraj P, Schafer PH, Cavender DE, Wu Y, Ngo K, Grealish PF, Wadsworth SA, Peterson PA, Siekierka JJ, Harris CA, and Fung-Leung WP. Interleukin (IL)-1 receptor-associated kinase (IRAK) requirement for optimal induction of multiple IL-1 signaling pathways and IL-6 production. J Exp Med 187: 2073–2079, 1998.[Abstract/Free Full Text]
16. Kawai T, Adachi O, Ogawa T, Takeda K, and Akira S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11: 115–122, 1999.[ISI][Medline]
17. Kloner RA and Jennings RB. Consequences of brief ischemia: stunning, preconditioning, and their clinical implications: part 1. Circulation 104: 2981–2989, 2001.[Abstract/Free Full Text]
18. Levine B, Kalman J, Mayer L, Fillit HM, and Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 323: 236–241, 1990.[Abstract]
19. Li M, Carpio DF, Zheng Y, Bruzzo P, Singh V, Ouaaz F, Medzhitov RM, and Beg AA. An essential role of the NF- B/Toll-like receptor pathway in induction of inflammatory and tissue-repair gene expression by necrotic cells. J Immunol 166: 7128–7135, 2001.[Abstract/Free Full Text]
20. Lomaga MA, Yeh WC, Sarosi I, Duncan GS, Furlonger C, Ho A, Morony S, Capparelli C, Van G, Kaufman S, van der Heiden A, Itie A, Wakeham A, Khoo W, Sasaki T, Cao Z, Penninger JM, Paige CJ, Lacey DL, Dunstan CR, Boyle WJ, Goeddel DV, and Mak TW. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev 13: 1015–1024, 1999.[Abstract/Free Full Text]
21. Macnicol MF, Goldberg AH, and Clowes GH Jr. Depression of isolated heart muscle by bacterial endotoxin. J Trauma 13: 554–558, 1973.[ISI][Medline]
22. McMurray JJ and Stewart S. Epidemiology, aetiology, and prognosis of heart failure. Heart 83: 596–602, 2000.[Free Full Text]
23. Medzhitov R, Preston-Hurlburt P, and Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388: 394–397, 1997.[Medline]
24. Muzio M, Natoli G, Saccani S, Levrero M, and Mantovani A. The human toll signaling pathway: divergence of nuclear factor kappaB and JNK/SAPK activation upstream of tumor necrosis factor receptor-associated factor 6 (TRAF6). J Exp Med 187: 2097–2101, 1998.[Abstract/Free Full Text]
25. Naito A, Azuma S, Tanaka S, Miyazaki T, Takaki S, Takatsu K, Nakao K, Nakamura K, Katsuki M, Yamamoto T, and Inoue J. Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 4: 353–362, 1999.[Abstract]
26. Nemoto S, Vallejo JG, Knuefermann P, Misra A, Defreitas G, Carabello BA, and Mann DL. Escherichia coli LPS-induced LV dysfunction: role of Toll-like receptor-4 in the adult heart. Am J Physiol Heart Circ Physiol 282: H2316–H2323, 2002.[Abstract/Free Full Text]
27. Niebauer J, Volk HD, Kemp M, Dominguez M, Schumann RR, Rauchhaus M, Poole-Wilson PA, Coats AJ, and Anker SD. Endotoxin and immune activation in chronic heart failure: a prospective cohort study. Lancet 353: 1838–1842, 1999.[ISI][Medline]
28. Ohashi K, Burkart V, Flohe S, and Kolb H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol 164: 558–561, 2000.[Abstract/Free Full Text]
29. Pagani FD, Baker LS, Hsi C, Knox M, Fink MP, and Visner MS. Left ventricular systolic and diastolic dysfunction after infusion of tumor necrosis factor- in conscious dogs. J Clin Invest 90: 389–398, 1992.[ISI][Medline]
30. Parker JL and Adams HR. Development of myocardial dysfunction in endotoxin shock. Am J Physiol Heart Circ Physiol 248: H818–H826, 1985.[Abstract/Free Full Text]
31. Parker MM, Shelhamer JH, Bacharach SL, Green MV, Natanson C, Frederick TM, Damske BA, and Parrillo JE. Profound but reversible myocardial depression in patients with septic shock. Ann Intern Med 100: 483–490, 1984.[ISI][Medline]
32. Parrillo JE, Burch C, Shelhamer JH, Parker MM, Natanson C, and Schuette W. A circulating myocardial depressant substance in humans with septic shock. Septic shock patients with a reduced ejection fraction have a circulating factor that depresses in vitro myocardial cell performance. J Clin Invest 76: 1539–1553, 1985.[ISI][Medline]
33. Poltorak A, He X, Smirnova I, Liu MY, Huffel CV, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, and Beutler B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085–2088, 1998.[Abstract/Free Full Text]
34. Schreiber E, Matthias P, Muller MM, and Schaffner W. Rapid detection of octamer binding proteins with `mini-extracts', prepared from a small number of cells. Nucleic Acids Res 17: 6419, 1989.[Free Full Text]
35. Schulz R, Panas DL, Catena R, Moncada S, Olley PM, and Lopaschuk GD. The role of nitric oxide in cardiac depression induced by interleukin-1 and tumour necrosis factor-. Br J Pharmacol 114: 27–34, 1995.[ISI][Medline]
36. Shames BD, Meldrum DR, Selzman CH, Pulido EJ, Cain BS, Banerjee A, Harken AH, and Meng X. Increased levels of myocardial IB- protein promote tolerance to endotoxin. Am J Physiol Heart Circ Physiol 275: H1084–H1091, 1998.[Abstract/Free Full Text]
37. Starr RG, Lader AS, Phillips GC, Stroman CE, and Abel FL. Direct action of endotoxin on cardiac muscle. Shock 3: 380–384, 1995.[ISI][Medline]
38. Stein B, Frank P, Schmitz W, Scholz H, and Thoenes M. Endotoxin and cytokines induce direct cardiodepressive effects in mammalian cardiomyocytes via induction of nitric oxide synthase. J Mol Cell Cardiol 28: 1631–1639, 1996.[ISI][Medline]
39. Suffredini AF, Fromm RE, Parker MM, Brenner M, Kovacs JA, Wesley RA, and Parrillo JE. The cardiovascular response of normal humans to the administration of endotoxin. N Engl J Med 321: 280–287, 1989.[Abstract]
40. Swantek JL, Cobb MH, and Geppert TD. Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for lipopolysaccharide stimulation of tumor necrosis factor alpha (TNF-) translation: glucocorticoids inhibit TNF-alpha translation by blocking JNK/SAPK. Mol Cell Biol 17: 6274–6282, 1997.[Abstract]
41. Swantek JL, Tsen MF, Cobb MH, and Thomas JA. IL-1 receptor-associated kinase modulates host responsiveness to endotoxin. J Immunol 164: 4301–4306, 2000.[Abstract/Free Full Text]
42. Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, Miyake K, Freudenberg M, Galanos C, and Simon JC. Oligosaccharides of hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med 195: 99–111, 2002.[Abstract/Free Full Text]
43. Thomas J, Tsen M, White DJ, and Horton JW. IRAK contributes to burn-triggered myocardial contractile dysfunction. Am J Physiol Heart Circ Physiol 283: H829–H836, 2002.[Abstract/Free Full Text]
44. Thomas JA, Allen JL, Tsen M, Dubnicoff T, Danao J, Liao XC, Cao Z, and Wasserman SA. Impaired cytokine signaling in mice lacking the IL-1 receptor-A associated kinase. J Immunol 163: 978–984, 1999.[Abstract/Free Full Text]
45. Thomas JA, Tsen MF, White DJ, and Horton JW. TLR4 inactivation and rBPI₂₁ block burn-induced myocardial contractile dysfunction. Am J Physiol Heart Circ Physiol 283: H1645–H1655, 2002.[Abstract/Free Full Text]
46. Torre-Amione G, Kapadia S, Benedict C, Oral H, Young JB, and Mann DL. Proinflammatory cytokine levels in patients with depressed left ventricular ejection fraction: a report from the Studies of Left Ventricular Dysfunction (SOLVD). J Am Coll Cardiol 27: 1201–1206, 1996.[Abstract]
47. Vig E, Green M, Liu Y, Donner DB, Mukaida N, Goebl MG, and Harrington MA. Modulation of tumor necrosis factor and interleukin-1-dependent NF-kappaB activity by mPLK/IRAK. J Biol Chem 274: 13077–13084, 1999.[Abstract/Free Full Text]
48. Wesche H, Henzel WJ, Shillinglaw W, Li S, and Cao Z. MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7: 837–847, 1997.[ISI][Medline]

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