Nuclear Factor-κB Decoy
Infiltrating the Heart of the Matter in Inflammatory Heart Disease
See related article, pages 899–906
Inflammation is part of the host response repertoire in defense against injury. Inflammatory processes play an important role in all the common cardiovascular diseases such as atherosclerosis, myocardial infarction, and heart failure. However, when the inflammatory process becomes uncontrolled, it can become part of the disease. This situation is best exemplified by inflammatory heart diseases such as viral or autoimmune myocarditis, where the immune system can be triggered by either external antigens such as a virus, or internal antigens such as myosin. The activated immune cells then undergo exuberant signal amplification, leading to clonal expansion of T lymphocytes and elaboration of cytokines, resulting in cell and matrix disruption and the phenotype of dilated cardiomyopathy. 1,2
Giant cell myocarditis is an example of disproportionate immune response, including coalescence of macrophages into giant cells mixed with lymphocytes, with associated high mortality and no specific treatment. To understand the pathogenesis of giant cell myocarditis, an experimental model of autoimmune myocarditis (EAM) induced by myosin antigen mixed with adjuvant in the murine model has been previously developed. 3,4 The myosin molecule contains a very specific epitope of approximately 8 amino acids in size that can also be mimicked by infectious agents, which is capable of triggering immune cell activation in an MHC-restricted context, leading to T cell–mediated destruction of the myocardium (Figure 1). This has been definitively demonstrated by humanized murine models in which the mouse MHC system has been substituted with the human homologues, and the disease can be perfectly reproduced with immunogens containing human myosin.5
The agents of destruction in this process include cytokines such as tumor necrosis factor (TNF)6 and nitric oxide produced in large quantities by the inducible nitric oxide synthase (iNOS).7 However, the immune system, being critical for host survival, is extremely redundant in its response. The question is whether there are additional central modulators of immune response that may be targets for therapeutic manipulation.
Potential Role of NF-κB as Immune Regulator
The activation of inflammatory response requires coordinated expression of a large number of effector systems to respond to external stress. The external stimuli, including infection, cytokine or neurohormone receptor activation, oxidative stress, matrix alterations, integrin binding, and cytoskeletal deformation, all converge through intracellular signaling pathways upon specific families of nuclear transcription factors, a prominent member being nuclear factor-κB (NF-κB).8
NF-κB is a dimeric transcription factor consisting of homodimers or heterodimers of Rel-related proteins. It normally resides in the cytoplasm, complexed with an inhibitor, IκB, and is in an inactive form. Upon activation by external stimuli, the inflammatory signals converge on a set of regulatory control IκB kinases known as the IKK complex. 9 The IKK complexes can phosphorylate IκB, leading to its ubiquitination and degradation by the proteosome (Figure 2). The liberated NF-κB then enters the nucleus, interacts with κB elements in the promoter region of many inflammatory response genes, and activates their transcription. NF-κB is thus one of the central regulators of inflammatory response.10 Additional signaling events, including phosphorylation of NF-κB, hyperphosphorylation of IKK, and transcriptional regulation of IκB and NF-κB, all modulate the level and duration of NF-κB activity. The classic antiinflammatory agent aspirin acts by binding IKKβ, thereby preventing the activation of NF-κB, similar to specific peptide strategies of NF-κB inhibition.11
The question is whether NF-κB also plays a central role in inflammatory heart disease, and whether it could represent a target for therapeutic modulation to alter the outcomes of the disease.
Role of NF-κB in Autoimmune Myocarditis: Insight Through Decoys
To test this strategy, Yokoseki and colleagues,12 in this issue of Circulation Research, have designed an NF-κB cis element decoy oligonucleotide (ODN), which was delivered in vivo using an HVJ-AVE-liposome system. The administered decoy ODN competes for the cis NF-κB binding site in the promoter region of several cytokine genes as demonstrated by gel-shift assays. The decoy ODN led to the reduced expression and production of ICAM-1, IL-2, TNF, and iNOS in the heart as demonstrated by real-time PCR and Western blotting. This treatment resulted in the reduction in the severity of EAM compared with the control mice by histopathology, heart to body weight ratios, and clinical outcome.
The decoy ODN competes with endogenous κB sites for binding to NF-κB (Figure 2). This decoy thus mimics, to a certain extent, the natural IκB inhibitors by complexing with the NF-κB transcription factor. However, it appears to work in the nucleus rather than the cytoplasm, as demonstrated by the nuclear localization of the administered complex. In turn, this strategy served as an important investigative tool to illustrate the central regulatory role played by nuclear factors such as NF-κB and its effector systems in the development of autoimmune myocarditis. Furthermore, this seems to be a more effective means of simultaneously modulating several immune response repertoires without having to completely disable a single immune response pathway.
NF-κB activation is likely very important for host survival, even though excessive activation can lead to exuberant inflammatory response that paradoxically induces host injury. In the setting of cytokine-induced intracellular activation where there is activation of both prosurvival signals and proapoptotic signals, NF-κB is responsible for most of the host survival factors. Thus, modulating NF-κB response rather than eliminating it will be an important part of the consideration in its targeting for therapeutic purposes.
The Potential of Molecular Decoys as Tools and Therapies
This decoy strategy of modulating the immune responses also opens the door for future targeted therapeutic manipulation of host immune response repertoire and has been reviewed previously in this journal.13 This approach has the advantage that it simultaneously modulated several effector pathways without singling out a particular pathway for complete elimination. This may help to preserve the overall immune response capability while reducing the overall intensity of inflammatory response that is responsible for the immune destruction of the myocardium in autoimmune myocarditis. NF-κB decoy has already shown potential benefit in models of tumor metastasis, cytokine-mediated apoptosis, and immune-mediated neural damage.
This strategy may also be applicable in other inflammation-related cardiovascular disease, including viral myocarditis, sepsis-induced cardiac depression, and even generic heart failure or atherosclerosis. However, whether NF-κB is the right target in these instances and how much and how long to treat for these conditions are yet to be determined.
It is important to keep in mind that the molecular decoys can sometimes have nonspecific effects in the target cells. Therefore having adequate controls, including scrambled ODNs, meticulous examination of changes in the nuclear transcription factors, and downstream gene transcription targets, will be critical to ensure that a “smart bomb” rather than a “shotgun” approach is achieved.
This work was supported in part by grants from the Heart and Stroke Foundation of Ontario and the Canadian Institutes of Health Research (CIHR). Dr Liu is the Heart & Stroke/Polo Chair Professor of Medicine at the University of Toronto.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
Liu P, Mason J. Advances in the understanding of myocarditis. Circulation. 2001; 104: 1076–1082.
Neu N, Rose NR, Beisel KW, Herskowitz A, Gurri-Glass G, Craig SW. Cardiac myosin induces myocarditis in genetically predisposed mice. J Immunol. 1987; 139: 3630–3636.
Bachmaier K, Neu N, Yeung RS, Mak TW, Liu P, Penninger JM. Generation of humanized mice susceptible to peptide-induced inflammatory heart disease. Circulation. 1999; 99: 1885–1891.
Bachmaier K, Pummerer C, Kozieradzki I, Pfeffer K, Mak TW, Neu N, Penninger JM. Low-molecular weight tumor necrosis factor receptor p55 controls induction of autoimmune heart disease. Circulation. 1997; 95: 655–661.
Zaragoza C, Ocampo C, Saura M, Leppo M, Wei XQ, Quick R, Moncada S, Liew FY, Lowenstein CJ. The role of inducible nitric oxide synthase in the host response to Coxsackievirus myocarditis. Proc Natl Acad Sci USA. 1998; 95: 2469–2474.
May MJ, D’Acquisto F, Madge LA, Glockner J, Pober JS, Ghosh S. Selective inhibition of NF-κB activation by a peptide that blocks the interaction of NEMO with the IκB kinase complex. Science. 2000; 289: 1550–1554.
Yokoseki O, Suzuki J-i, Kitabayashi H, Watanabe N, Wada Y, Aoki M, Morishita R, Kaneda Y, Ogihara T, Futamatsu H, Kobayashi Y, Isobe M. cis Element decoy against nuclear factor-κB attenuates development of experimental autoimmune myocarditis in rats. Circ Res. 2001; 89: 899–906.
Morishita R, Higaki J, Tomita N, Ogihara T. Application of transcription factor “decoy” strategy as means of gene therapy and study of gene expression in cardiovascular disease. Circ Res. 1998; 82: 1023–1028.