Interleukin-18 as a Therapeutic Target in Acute Myocardial Infarction and Heart Failure

INTRODUCTION

Heart failure (HF) is a clinical syndrome of impaired left ventricular function characterized by shortness of breath, fatigue and poor exercise tolerance (1). In 2010, hospital discharges for HF in the United States were estimated to be one million, and one in nine deaths had HF mentioned on the death certificate (2). Patient survival has improved in recent years, but the death rate remains unacceptably high, with approximately 50% of people diagnosed with HF dying within five years (2). Inflammation is a central component of the response to tissue stress and injury in the heart, coordinating remodeling and healing by promoting extracellular matrix remodeling, cell proliferation, cardiomyocyte hypertrophy and affecting cardiomyocyte contractility (3). While some inflammation is necessary for proper healing, increased inflammation appears to play a role in the predisposition to develop heart disease and may contribute to the disease severity and response to treatment (4–6). Increased inflammatory biomarkers correlate with HF severity and predict adverse prognosis (1,4,7). In experimental settings, the administration of proinflammatory cytokines promotes left ventricular dysfunction (1,8). However, antiinflammatory strategies in the treatment of HF are currently lacking, indicating that the inflammatory mechanisms involved in the development of HF are incompletely characterized.

INTERLEUKIN-18, AN IL-1 FAMILY MEMBER

Interleukin-18 (IL-18) is a proinflammatory cytokine that was first described in 1989 for its ability to induce interferon γ (IFN-γ) production (9). The cytokine was later cloned and found to have a synergistic effect with IL-12 in the production of IFN-γ from T cells, natural killer (NK) cells and macrophages (10,11). This synergism is thought to be the result of IL-12 inducing the expression of the IL-18 receptor on T cells (12). IL-18 stimulates the proliferation of T cells, making it functionally related to IL-12, but it is most similar structurally to the IL-1 family of cytokines, specifically IL-1β (11,13) (Table 1). Because of this structural similarity and other common characteristics shared with IL-1β, IL-18 is part of the IL-1 family. Like IL-1β, IL-18 has a secondary structure primarily consisting of β-sheets (11).

IL-1β and IL-18 both are activated by caspase-1 following formation of the inflammasome. The inflammasome is a macromolecular structure consisting of (a) an intracellular NOD-like receptor (NLR) such as NLRP3, (b) an adaptor protein, apoptosis speck-like protein containing a caspase recruitment domain (ASC), and (c) procaspase-1 (14,15) (Figure 1). Accumulation of adenosine triphosphate (ATP) and cell debris after tissue injury, among other stimuli, acts as a danger signal and induces formation of the inflammasome resulting in the cleavage and activation of caspase-1, which subsequently cleaves the inactive precursor proteins pro-IL-1β and pro-IL-18 (16,17) (see Figure 1). Unlike pro-IL-1β, pro-IL-18 is constitutively expressed in unstimulated cells (18). Activation of the inflammasome occurs in different cell types in response to injury (19). After acute myocardial infarction (AMI), the inflammasome is formed in leukocytes, endothelial cells, fibroblasts and cardiomyocytes (15,20,21). Although following AMI, active IL-1β and IL-18 are increased in the ischemic myocardial tissue, in vitro cell studies have evidenced a different function of the inflammasome in the different cell types (22). Leukocytes produce much IL-1β and IL-18 and inflammasome positive leukocytes are found in the infarct area (15). In fibroblasts, the activation of the inflammasome represents a stimulus for myofibroblast differentiation and collagen synthesis by increasing the local production of IL-1β and IL-18 (21,23). In cardiomyocytes, the activation of the inflammasome leads to caspase-1 activation and pyroptosis, but not to IL-1β release.

Also, pro-IL-1β and pro-IL-18 can be activated by extracellular neutrophil proteinases such as neutrophil proteinase 3 (PR3) during tissue injury (24,25). Furthermore, pro-IL-18 can be cleaved by human chymase from mast cells at two sites, creating two unique species, only one of which is biologically active, p16 (26). The p16 fragment-induced IFN-γ production in vitro but with only approximately 20% of the activity of mature IL-18 (26). Adding supernatant from human CD8⁺ T cells containing granzyme B (GrB) resulted in cleavage at the same site as caspase-1 and resulted in active IL-18 (27). The proteases elastase and cathepsin G cleave and activate IL-1β and have been suggested to cleave IL-18 as well in human neutrophils, however this cleavage does not result in biologically active species (28,29). Although most IL-18 is soluble, IL-18 can exist as a membrane-bound protein in a subset of macrophages. Upon stimulation with lipopolysaccharide (LPS), these cells secrete soluble IL-18, suggesting an additional mechanism of IL-18 release and activation by a protease induced by LPS, possibly PR3 (30,31). PR3 could also be the mechanism by which pro-IL-1β and pro-IL-18 released from a dying cell are activated. Conversely, IL-18 is inactivated through cleavage by caspase-3 (32).

INTERLEUKIN-18 SIGNALING

Similar to the receptors for IL-1 and other cytokines, the IL-18 receptor (IL-18R) is a dimer composed of IL-18Rα, a low affinity binding chain, and IL-18Rβ (also known as accessory protein-like [AcPL]) that binds the IL-18/IL-18Rα complex (33,34) (see Figure 1). Both receptor subunits are members of the IL-1 receptor family; IL-18Rα is also known as IL-1 receptor related protein (IL-1rp) (35). IL-37, on the other hand, binds IL-18Rα, but does not recruit IL-18Rβ, and instead recruits TIR8, also known as single immunoglobulin interleukin-1 receptor–related molecule (SIGIRR), containing a Toll/IL-1R (TIR) domain (see Figure 1) (36–38). Since an active receptor complex is not formed, there is no signaling through MyD88. Once the active receptor complex is assembled, IL-18R shares many downstream signaling pathways with the IL-1 receptor (39). Both the IL-1 and IL-18 receptors can recruit IL-1 receptor-associated kinase (IRAK) and MyD88, an intracellular adaptor molecule required for IRAK phosphorylation and for IRAK interaction with the receptor. Mice lacking MyD88 have decreased T cell proliferation and production of inflammatory cytokines in response to IL-1 and impaired IFN-γ, nuclear factor κB (NFκB), and c-Jun N-terminal kinase (JNK) signaling in response to IL-18 (40). Similar signaling disruptions were seen when IL-18 was given to IRAK-deficient helper T cell type 1 (Th1) cells and IRAK knockout mice, suggesting an important role for IRAK in IL-18 signaling (41). Like the IL-1 receptor, the IL-18R recruits tumor necrosis factor receptor–associated factor-6 (TRAF6) which allows for the activation of NF-κB and its translocation to the nucleus (42) (see Figure 1). IL-18 also activates p38 mitogen-activated protein kinase (MAPK) (43). A direct comparison of IL-1β and IL-18 signaling showed that IL-18 preferentially activates p38-MAPK and has minimal effects on the induction of cyclooxygenase-2 (COX-2) which mediates fever through synthesis of prostaglandin E2, whereas IL-1β induces a rapid and intense COX-2 expression (39).

In the search for a soluble IL-18 receptor, an IL-18 binding protein (IL-18BP) was found by running samples of human urine through an IL-18 agarose column (44). IL-18BP is able to block the activity of both human and murine IL-18 and prevent LPS-induced IFN-γ production in mouse splenocytes (44). IL-18BP is not a member of the IL-1 or IL-18 receptor families, and has six naturally occurring isoforms (44,45) (Figure 2). Human IL-18BPa (hIL-18BPa) has the greatest affinity for IL-18, and adding supernatants from cells expressing this protein to human and murine spleen cells resulted in a 90% decrease and 80% decrease in IL-18 activity, respectively (45). Human IL-18BPc (hIL-18BPc) and murine IL-18BPd (mIL-18BPd) show similar results, while murine IL-18BPc (mIL-18BPc) is specific for murine IL-18, and human IL-18BPb (hIL-18BPb) and IL-18BPd (hIL-18BPd) lack the complete Ig domain and therefore cannot bind IL-18 (45).

INTERLEUKIN-18 LEVELS IN PATIENTS WITH HEART DISEASE

There is growing evidence for a role of IL-18 in human myocardial infarction, HF and other forms of heart disease. IL-18 expression is increased in human atherosclerotic plaques collected during carotid endarterectomy and its accumulation is associated with plaque destabilization (46). A recent study conducted in the community-based cohort of Framingham, however, reported no significant correlations between the levels of IL-18 and subclinical aortic atherosclerosis (47). High serum levels of IL-18 were associated with an increased risk of developing cardiovascular disease (CVD) in the general population, increased mortality in HF patients and development of congestive HF and AMI in patients with acute coronary syndromes (48–52).

Plasma IL-18 levels also were increased in patients with AMI compared with aged matched controls and correlated with increased atrial natriuretic peptide (ANP) levels, suggesting that IL-18 may play a role in inducing ANP (53). Immunohistochemistry revealed increased IL-18 and IL-18Rα expression in endothelial cells, macrophages and cardiomyocytes and decreased IL-18BP in the myocardium of patients with end-stage HF undergoing heart transplantation (54). Parallel to increased levels of IL-18, an increase in IL-18BP levels is seen in many disease processes. The naturally occurring IL-18BP in the circulation neutralizes the circulating IL-18 so that the “free” IL-18 levels are much lower than the total IL-18 levels, and the “free” IL-18 levels seem to better correlate with disease activity than the total IL-18 levels (55).

INTERLEUKIN-18 IN IN VIVO AND IN VITRO MODELS

CONCLUSION AND FUTURE DIRECTIONS

Over the last 20 years, IL-18 has proven to be much more than simply an IFN-γ inducing factor. An increase in IL-18 activity has been correlated with a number of human pathologies including AMI, HF, and pressure-overload. Considering the role of IL-18 in mediating acute cardiac effects such as contractile dysfunction and β-AR desensitization as well as chronic cardiac changes like hypertrophy and fibrosis, it is reasonable to hypothesize that IL-18 blockade may represent a valuable strategy in both acute and chronic cardiac diseases (Figure 4). Genetic deletion or neutralization of IL-18 reduces myocyte hypertrophy in models of pressure overload or LPS-induced myocardial dysfunction (60,72). Preventing IL-18 activity by pretreatment with a neutralizing antibody, IL-18BPa, NLRP3 inhibitors or caspase-1 inhibitors improves contractile function and decreases infarct size after ischemia-reperfusion injury, providing the basis for additional studies of these molecules in AMI (63,71,90). Inhibition of IL-18 may, however, impede the physiologic hypertrophic response following pressure overload, and lead to maladaptive remodeling (53). Preclinical studies of IL-18 targeted treatments with IL-18BP or IL-18Ab given during ischemia or at the time of reperfusion are lacking. If such studies were able to confirm a protective effect of IL-18 that is maintained over time and not associated with an impairment in infarct healing in preclinical AMI modes, then pilot clinical trials in ST-segment elevation AMI (STEMI) and non-STEMI may be warranted, as has occurred with IL-1 blockers (92–95). A comparison between IL-18 and IL-1 blockers in AMI would provide further understanding of whether the two cytokines have overlapping or synergistic effects or whether IL-18 mediates IL-1 effects (as suggested in [74]). The acute cardiodepressant effect of IL-18 suggests that IL-18 blockade may represent a valuable treatment for acute decompensated or chronic symptomatic HF. The effects of IL-18 appear to affect both systolic and diastolic function (51,52). Preliminary data with IL-1 blockade show that IL-1 activity may be a modifiable factor in patients with HF (1,3,94,96,97) and the mechanistic links between IL-18 and IL-1 suggest that IL-18 blockade may have similar beneficial effects (1,74). Therefore IL-18 blockade may be used in those patients with elevated IL-1 to improve LV function without altering other IL-1 mediated effects.

Heart failure with preserved ejection fraction (HFPEF) is a disease characterized by impaired filling of the LV due to increased fibrosis and cardiomyocyte hypertrophy (98). Inflammatory cytokines contribute to the pathophysiology of HFPEF (96,99), thus IL-18 is a potential target for HFPEF due to its prohypertrophic and profibrotic effects (56–60,84,86,87). The chronic prohypertrophic and profibrotic effects of IL-18 in animal studies suggest that a longer duration of IL-18 blockade may prevent, or even reverse, the development of pathologic cardiac hypertrophy; however it also may prevent development of physiologic hypertrophy (53).

Targeted intervention of IL-18 might be advantageous considering the differences in signaling with IL-1 on COX-2 induction (39). The effects of IL-18 blockade on the host defense against opportunistic infections in patients will, however, need to be assessed (100–102). The decision to proceed with one or more pilot studies in this area will likely depend on the safety and pharmacokinetic profiles of the agents. Short-acting agents (for example, r-hIL-18BP) may be advantageous for conditions in which a rapid on/rapid off effect is desired and a short-term treatment is envisioned; whereas long-acting agents (for example, an IL-18 neutralizing antibody) will likely serve well for conditions in which longer duration of treatment is necessary. Many antiinflammatory strategies found effective in the animal models of heart disease have, however, failed to show beneficial effects in clinical trials, and therefore caution should be used when extrapolating preclinical results to clinical use (3).