Role of TLR-2 in the Activation of Nuclear Factor κB by Oxidative Stress in Cardiac Myocytes

Growing evidence from patients with heart failure and from experimental animal models implicates effectors of innate immunity in the pathogenesis of this syndrome. The expression of the innate immunity signaling protein, Toll-like receptor 4 (TLR4), is increased in cardiac myocytes in situ and in failing myocardium, but the mechanism by which TLRs may be activated in the failing heart remains unclear. We report that TLR2, which is expressed in cardiac myocytes, participates in the response of these cells to oxidative stress, a major contributor to the pathogenesis of cardiac dysfunction. Hydrogen peroxide increased nuclear factor κB (NF-κB) activation in Chinese hamster ovary fibroblasts that overexpress TLR2 but not in normal or TLR4-overexpressing Chinese hamster ovary cells, an effect that was abrogated by an α-TLR2 antibody. In neonatal rat ventricular myocytes, the α-TLR2 antibody inhibited hydrogen peroxide-induced nuclear translocation of NF-κB and activator protein-1 (AP-1). Inhibition of TLR2 had no effect on tumor necrosis factor α-induced NF-κB or AP-1 activation, on the DNA binding of the basal transcription factor Oct-1, or on hydrogen peroxide-induced phosphorylation of p38 MAP kinase. Importantly, oxidative stress-induced cytotoxicity was enhanced by blocking TLR2. Given the importance of cytotoxicity and apoptosis to the pathology of the ischemic heart, an anti-apoptotic effect of TLR2 in cardiac myocytes exposed to elevated levels of ROS may limit further cardiac dysfunction. Growing evidence from patients with heart failure and from experimental animal models implicates effectors of innate immunity in the pathogenesis of this syndrome. The expression of the innate immunity signaling protein, Toll-like receptor 4 (TLR4), is increased in cardiac myocytes in situ and in failing myocardium, but the mechanism by which TLRs may be activated in the failing heart remains unclear. We report that TLR2, which is expressed in cardiac myocytes, participates in the response of these cells to oxidative stress, a major contributor to the pathogenesis of cardiac dysfunction. Hydrogen peroxide increased nuclear factor κB (NF-κB) activation in Chinese hamster ovary fibroblasts that overexpress TLR2 but not in normal or TLR4-overexpressing Chinese hamster ovary cells, an effect that was abrogated by an α-TLR2 antibody. In neonatal rat ventricular myocytes, the α-TLR2 antibody inhibited hydrogen peroxide-induced nuclear translocation of NF-κB and activator protein-1 (AP-1). Inhibition of TLR2 had no effect on tumor necrosis factor α-induced NF-κB or AP-1 activation, on the DNA binding of the basal transcription factor Oct-1, or on hydrogen peroxide-induced phosphorylation of p38 MAP kinase. Importantly, oxidative stress-induced cytotoxicity was enhanced by blocking TLR2. Given the importance of cytotoxicity and apoptosis to the pathology of the ischemic heart, an anti-apoptotic effect of TLR2 in cardiac myocytes exposed to elevated levels of ROS may limit further cardiac dysfunction. toll-like receptor interleukin-1 c-Jun NH2-terminal kinase lipopolysaccharide activator protein-1 tumor necrosis factor Chinese hamster ovary neonatal rat ventricular myocyte reverse transcriptase-polymerase chain reaction base pair(s) phosphate-buffered saline fluorescein isothiocyanate fluorescence-activated cell sorting electrophoretic mobility shift assay mitogen-activated protein kinase phosphatidylserine antibody pattern recognition receptor reactive oxygen species heat shock protein The mammalian toll-like receptors (TLRs)1 are a recently recognized family of proteins implicated in directing innate immune responses by components of diverse pathogens (1Medzhitov R. Janeway Jr., C. N. Engl. J. Med. 2000; 343: 338-344Crossref PubMed Scopus (1736) Google Scholar, 2Schuster J.M. Nelson P.S. J. Leukoc. Biol. 2000; 67: 767-773Crossref PubMed Scopus (91) Google Scholar). So far, six human TLRs have been cloned (3Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1451) Google Scholar, 4Takeuchi O. Kawai T. Sanjo H. Copeland N.G. Gilbert D.J. Jenkins N.A. Takeda K. Akira S. Gene. 1999; 231: 59-65Crossref PubMed Scopus (353) Google Scholar), and several others are suggested to be present. Medzhitov et al. (5Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4437) Google Scholar) were the first to show that constitutively active TLR4 induces the activation of NF-κB with the subsequent expression of IL-1, IL-6, IL-8, and the costimulatory molecule B7.1. Active TLR4 can also induce c-Jun NH2-terminal kinase (JNK) (6Muzio M. Natoli G. Saccani S. Levrero M. 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The most convincing evidence exists for a role of TLR4 in the recognition of bacterial LPS (lipopolysaccharides). Mice with either a spontaneous mutation of the TLR4 gene or targeted disruption of the gene have no response to lipopolysaccharides and are thus resistant to endotoxic shock (11Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar, 12Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar, 13Qureshi S.T. Lariviere L. Leveque G. Clermont S. Moore K.J. Gros P. Malo D. J. Exp. Med. 1999; 189: 615-625Crossref PubMed Scopus (1354) Google Scholar). In contrast, mice with targeted disruption of TLR2 have no deficit in the recognition of Gram-negative bacteria (14Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2790) Google Scholar). Interestingly, missense mutations affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS in humans (15Arbour N.C. Lorenz E. Schutte B.C. Zabner J. Kline J.N. Jones M. Frees K. Watt J.L. Schwartz D.A. Nat. Genet. 2000; 25: 187-191Crossref PubMed Scopus (1785) Google Scholar). Most studies have focused on TLR expression and function in mononuclear cells and monocyte-like cell lines; however, TLRs differentially exhibit a wider pattern of tissue expression, including the lung, heart, and brain (2Schuster J.M. Nelson P.S. J. Leukoc. Biol. 2000; 67: 767-773Crossref PubMed Scopus (91) Google Scholar). Thus, TLRs may additionally serve as pro-inflammatory receptors in cell types without a dedicated immune function. TLR4, for example, is expressed by human dermal endothelial cells and can activate NF-κB (16Zhang F.X. Kirschning C.J. Mancinelli R. Xu X.P. Jin Y. Faure E. Mantovani A. Rothe M. Muzio M. Arditi M. J. Biol. Chem. 1999; 274: 7611-7614Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). We previously demonstrated (17Frantz S. Kobzik L. Kim Y.D. Fukazawa R. Medzhitov R. Lee R.T. Kelly R.A. J. Clin. Invest. 1999; 104: 271-280Crossref PubMed Scopus (564) Google Scholar) that TLR4 is expressed in the heart and that injured human and murine myocardium exhibit focal areas of intense TLR4 expression. The reason for this differential expression of TLR4, as well as the function of TLR4 in the injured heart, in the absence of infection, remains unknown. Although the identity of endogenous ligands, analogous toDrosophila spatzle, for the vertebrate TLRs remains elusive, a recent report suggests that the stress-associated factor heat-shock protein 60 signals through TLR4 (18Ohashi K. Burkart V. Flohe S. Kolb H. J. Immunol. 2000; 164: 558-561Crossref PubMed Scopus (1368) Google Scholar). Thus, innate immunity receptors may not only be activated by microorganisms, but also by endogenous signals that originate from injured cells that emanate “danger signals” (19Matzinger P. Semin. Immunol. 1998; 10: 399-415Crossref PubMed Scopus (631) Google Scholar). Oxidative stress in the post-ischemic heart has been identified as an important event in myocardial dysfunction, linked to both myocardial hypertrophy and apoptosis. Oxidative stress is known to induce cell death by apoptotic and necrotic pathways in isolated cardiac myocytes (20von Harsdorf R. Li P.F. Dietz R. Circulation. 1999; 99: 2934-2941Crossref PubMed Scopus (515) Google Scholar) and to trigger pro-inflammatory signaling pathways that activate NF-κB and AP-1 transcription factors (21Bowie A. O'Neill L.A. Biochem. Pharmacol. 2000; 59: 13-23Crossref PubMed Scopus (818) Google Scholar, 22Peng M. Huang L. Xie Z.J. Huang W.H. Askari A. Cell Mol. Biol. Res. 1995; 41: 189-197PubMed Google Scholar). Numerous mechanisms are suggested to underlie these effects of hydrogen peroxide, which are likely to be cell type-specific. Herein, we show a requirement of TLR2 for the activation of NF-κB and AP-1 by hydrogen peroxide and provide evidence that TLR2 imparts an anti-apoptotic effect in stressed or injured cardiac myocytes. Human recombinant TNFα was purchased from Endogen (Woburn, MA). All other chemicals, including hydrogen peroxide, were purchased from Sigma Chemical Co. unless noted otherwise. Chinese hamster ovary K1 (CHO-K1) fibroblasts were obtained from D. T. Golenbock (23Lien E. Sellati T.J. Yoshimura A. Flo T.H. Rawadi G. Finberg R.W. Carroll J.D. Espevik T. Ingalls R.R. Radolf J.D. Golenbock D.T. J. Biol. Chem. 1999; 274: 33419-33425Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar). The cell lines stably express CD-14, an NF-κB-dependent CD25 reporter construct, and empty vector (CHO/control), human TLR2 (CHO/TLR2), or human TLR4 (CHO/TLR4). The cells were maintained as described (23Lien E. Sellati T.J. Yoshimura A. Flo T.H. Rawadi G. Finberg R.W. Carroll J.D. Espevik T. Ingalls R.R. Radolf J.D. Golenbock D.T. J. Biol. Chem. 1999; 274: 33419-33425Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar). Cells were stimulated as indicated in Ham's F-12 medium containing 10% fetal calf serum (Life Technologies, Inc.). Neonatal rat ventricular myocytes (NRVM) were isolated from 1-day-old Harlan Sprague-Dawley pups as described (17Frantz S. Kobzik L. Kim Y.D. Fukazawa R. Medzhitov R. Lee R.T. Kelly R.A. J. Clin. Invest. 1999; 104: 271-280Crossref PubMed Scopus (564) Google Scholar). NRVM were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Life Technologies, Inc.). After 48 h, the medium was changed to Dulbecco's modified Eagle's medium /F-12 medium (Life Technologies, Inc.) containing 1% insulin, transferrin, selenium media supplement (ITS, Sigma) with antibiotics. Treatments were initiated 12 h later. CHO fibroblasts and NRVM were pretreated with a TLR-2 blocking antibody raised to a peptide that maps to the amino terminus of human TLR2 (5 or 10 μg/ml, number N-17, Santa Cruz Biotechnologies Inc., Santa Cruz, CA) for 1 h prior to addition of experimental treatments. Expression of rat TLR 1–6 were assessed using total RNA by RT-PCR from NRVM after treatment with deoxyribonuclease 1 (Life Technologies, Inc.) as described in the manufacturer's protocol. cDNA was generated using Superscript II (Life Technologies, Inc.). As a negative control, no reverse transcriptase was added. PCR primers were for TLR1 (383 bp) 5′-AAA CGG TCT CAT CCA CGT TC and 5′-GAG CAA TTG GCA GCA CAC TA, for TLR bp) CAA and GCA for bp) and for TLR4 and the bp) CAT and for bp) CCA and and for bp) and CAA T. All PCR were by a and DNA were by the with were in Cells were with in phosphate-buffered saline for 10 and exposed for 10 to in After to in cells were with normal serum Cells were by the of the human TLR2 an a rat antibody and a antibody were with a The of the antibody was assessed by the antibody with the peptide Cruz Biotechnologies Inc.). cells were in 10% After cell were of the protein were on a or and to After blocking with in saline with were with or p38 After several the were for 1 h with a antibody After were with a and CHO cells were stimulated as indicated in Ham's F-12 medium with 10% fetal calf serum for Cells were with and with an CD25 antibody was on using a Cells were as indicated for h with the blocking antibody for 1 shift were as described T. in Scholar). were with 10 of nuclear The binding for AP-1 an reaction a of and were with nuclear as DNA were on a in and were purchased from Santa Cruz was to cells with a of DNA and expression of phosphatidylserine by with V. NRVM were by with cells, and with After with cells were with a containing (5 for NRVM in the were identified using and are expressed as the of cells in of NRVM were by and with to the manufacturer's was on using a All are expressed as and of In with of an was In with of was by was were using Inc., human TLR have been identified (3Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1451) Google Scholar, 4Takeuchi O. Kawai T. Sanjo H. Copeland N.G. Gilbert D.J. Jenkins N.A. Takeda K. Akira S. Gene. 1999; 231: 59-65Crossref PubMed Scopus (353) Google Scholar). in cardiac tissue is unknown. TLR2, TLR4, and were by reverse in NRVM 1 for TLR1 and were in cardiac the TLR1 and not We previously that TLR4 protein is expressed by cardiac myocytes (17Frantz S. Kobzik L. Kim Y.D. Fukazawa R. Medzhitov R. Lee R.T. Kelly R.A. J. Clin. Invest. 1999; 104: 271-280Crossref PubMed Scopus (564) Google Scholar) but not the expression of TLR2. a TLR2 a was in a cell but not in or TLR4-overexpressing cell be in of neonatal cardiac myocytes 1 with the antibody the expression of TLR2 in myocytes of neonatal rat ventricular that was inhibited by with the TLR2 peptide to the antibody. cells also expressed identity as cardiac myocytes. oxygen a role in cardiac ischemic a role of TLRs in the response to oxidative stress, CHO cell lines were CHO fibroblasts not express for and TLR2 H. Kirschning C. E. B. Rothe M. Golenbock D. J. Immunol. 1999; 162: Google Scholar). The obtained cell lines have previously been (23Lien E. Sellati T.J. Yoshimura A. Flo T.H. Rawadi G. Finberg R.W. Carroll J.D. Espevik T. Ingalls R.R. Radolf J.D. Golenbock D.T. J. Biol. Chem. 1999; 274: 33419-33425Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar) and overexpress CD25 the of an NF-κB and empty vector (CHO/control), human TLR2 (CHO/TLR2), or human TLR4 (CHO/TLR4). Oxidative stress was by the of hydrogen peroxide The of NF-κB be by expression of CD25 by cells expression of TNFα increased CD25 expression in the and lines after h of of hydrogen peroxide increased NF-κB in the cells to a in the and cell lines In hydrogen peroxide increased nuclear translocation of NF-κB in the but was in the after h of as by was no in the of nuclear NF-κB for cells a with a TLR2 blocking antibody but not not abrogated the nuclear translocation of The binding of Oct-1, a transcription factor that is constitutively was by the treatment CHO cells a basal of nuclear AP-1 by without and was by with the TLR2 antibody or treatment with hydrogen peroxide not that TLR2 hydrogen peroxide-induced activation of TLR2 participates in the response to oxidative stress in cardiac myocytes, NRVM were with the blocking TLR2 antibody for 1 h and with hydrogen peroxide for As for the CHO cells, the TLR2 blocking antibody the nuclear translocation of NF-κB with was without NF-κB activation by TNFα was by treatment with the TLR2 blocking antibody. to AP-1 by hydrogen peroxide be by the TLR2 blocking but not AP-1 by The constitutively expressed was by the treatments. Oxidative stress is also known to induce the phosphorylation of p38 in cardiac myocytes Liu Y. M. Xu J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). blocking of TLR2 had no effect on hydrogen peroxide-induced p38 phosphorylation after of treatment NF-κB activation can anti-apoptotic and pathways in cell NF-κB or apoptosis on the of cell and the of M. 1999; PubMed Scopus Google Scholar). the of ischemic and oxidative stress, NF-κB to have an anti-apoptotic role in cardiac myocytes S. A. D. L.A. J. 2000; PubMed Google Scholar). the role of TLR2 in myocyte after oxidative stress, NRVM were with a blocking TLR2 antibody for 1 h and with hydrogen The of myocytes with DNA as the of cells in from to the Hydrogen peroxide increased this number to 12 to a of with the TLR2 antibody further increased hydrogen peroxide-induced DNA to a of was without effect on the response to hydrogen has a for phosphatidylserine after cells are from the to the of the cell thus a of cells D.M. J. Exp. Med. 1995; PubMed Scopus Google Scholar). levels of cells in these were to be Hydrogen peroxide had no effect but increased of NRVM after h to As in 4 of TLR2 increased the response to hydrogen peroxide and h with a was without Thus, of TLR2 hydrogen peroxide-induced NF-κB and AP-1 but hydrogen peroxide-induced DNA and in an anti-apoptotic function of TLR2 in cardiac myocytes after oxidative The innate immunity is an and of the immune that is by and recognition In the of innate have to and innate immunity by of but not by Jr., C.A. Immunol. Full Text PDF PubMed Scopus Google Scholar, R. Janeway Jr., C.A. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). pattern be bacterial lipopolysaccharides for (19Matzinger P. Semin. Immunol. 1998; 10: 399-415Crossref PubMed Scopus (631) Google Scholar, P. Immunol. PubMed Scopus Google Scholar) has suggested a for function and of the innate immune In the the immune be activated by endogenous or which originate from or necrotic cells and not by in signals are recognized by the pattern recognition receptors and activate the innate immune of the innate immune or that or cells in the absence of of cells be by in the expressed on the of cells or by the of proteins not in the cell myocardial expression of as and IL-6, in human and experimental heart of and without evidence of S. K. D. Y. B. H. N. Clin. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Although is a that or of these proteins to cardiac the that trigger and expression are not well of these is known to be to innate and expression may be initiated through activation of innate immunity We have previously that TLR4 is expressed in cardiac myocytes and that of TLR4 In normal murine and human myocardium, TLR4 expression was and expressed in cardiac myocytes. in both murine and human myocardium from of ischemic and in heart tissue from patients with heart focal areas of intense TLR4 in myocytes be (17Frantz S. Kobzik L. Kim Y.D. Fukazawa R. Medzhitov R. Lee R.T. Kelly R.A. J. Clin. Invest. 1999; 104: 271-280Crossref PubMed Scopus (564) Google Scholar). pattern recognition as for may be important to trigger activation of the innate immune in heart failure and may have an important In this we provide evidence for a function of the TLR2 which is also expressed in cardiac myocytes. oxygen species are to be a major factor for and ischemic in the oxidative stress in hydrogen peroxide was in neonatal rat ventricular myocytes. lines of evidence are that role in the nuclear translocation of NF-κB after with hydrogen hydrogen peroxide increased NF-κB and nuclear translocation in CHO cells that overexpress TLR2 but not in CHO cells that a TLR2 or that overexpress an but not a abrogated hydrogen peroxide-induced activation of NF-κB and AP-1 in CHO cells and effect of the TLR2 a was in NRVM that to a in cell NF-κB and AP-1 was by the DNA binding of the transcription factor was and hydrogen peroxide-induced phosphorylation of p38 was by the TLR2 antibody. 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