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ST2 pathway, inflammation and atherosclerosis: Trigger and target?
Accepted Manuscript The IL-33/ST2 pathway, inflammation and atherosclerosis: Trigger and target?
Alberto Aimo, Paola Migliorini, Giuseppe Vergaro, Maria Franzini, Claudio Passino, Alan Maisel, Michele Emdin PII: DOI: Reference:
S0167-5273(18)30957-4 doi:10.1016/j.ijcard.2018.05.056 IJCA 26469
To appear in: Received date: Revised date: Accepted date:
8 February 2018 19 April 2018 17 May 2018
Please cite this article as: Alberto Aimo, Paola Migliorini, Giuseppe Vergaro, Maria Franzini, Claudio Passino, Alan Maisel, Michele Emdin , The IL-33/ST2 pathway, inflammation and atherosclerosis: Trigger and target?. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ijca(2017), doi:10.1016/j.ijcard.2018.05.056
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ACCEPTED MANUSCRIPT The IL-33/ST2 Pathway, Inflammation and Atherosclerosis: Trigger and Target?
Alberto Aimo, MD,1,2 Paola Migliorini, MD, PhD,3 Giuseppe Vergaro, MD, PhD,1,4 Maria Franzini,
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PhD,5 Claudio Passino, MD,1,4 Alan Maisel, MD,6 Michele Emdin, MD, PhD1,4
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Author affiliations:
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2. Cardiology Division, University of Pisa, Italy
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1. Institute of Life Sciences, Scuola Superiore Sant’Anna, Pisa, Italy
3. Allergy and Immunology Division, University of Pisa, Pisa, Italy
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4. Cardiology Division, Fondazione Toscana Gabriele Monasterio, Pisa, Italy 5. Clinical Pathology Unit, University of Pisa, Italy
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6. University of California, San Diego, CA, USA.
Conflicts of interest: A.M. is consultant for Critical Diagnostics
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Word counts: 2511 (text)
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Address for correspondence: Alberto Aimo, MD
Scuola Superiore Sant'Anna and Cardiology Division, University of Pisa Piazza Martiri della Libertà, 33, Pisa, Italy Tel +39 050883111 Fax +39 050883225 Phone +39 3477084391 E-mail: [email protected], [email protected] 1
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Abstract The “inflammatory hypothesis” of atherosclerosis postulates that inflammatory cell signalling drives the formation, growth and ultimately the instability of atherosclerotic plaques, setting up the
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substrate for the thrombotic response that causes myocardial damage or infarction. The recent
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Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) trial has been hailed as
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the first demonstration, ex iuvantibus, of the inflammatory hypothesis. Indeed, interleukin (IL)-1β inhibition was found to reduce cardiovascular events in patients with previous myocardial infarction
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and raised high-sensitivity C-reactive protein, despite no effects on the lipid profile. These results prompt a dissection of inflammatory mechanisms of atherosclerosis in order to search for specific
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biomarkers with prognostic value and/or therapeutic targets.
Under this respect, the IL-33/suppression of tumorigenesis 2 (ST2) pathway deserves
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consideration. Indeed, its elements are particularly expressed in the endothelium of arterial vessels,
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and the interaction between IL-33 and the ST2 receptor blunts the immune response characteristic of atherosclerosis. By contrast, soluble ST2 (sST2) acts as a decoy receptor for IL-33, thus blocking
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its protective effects. Despite a solid theoretical framework, no definite demonstration of an involvement of the IL-33/ST2 pathway in atherosclerosis has been provided. Therefore, further
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studies are warranted to verify if elements of the IL-33/ST2 pathway may be proposed as markers of plaque burden and predictors of future cardiovascular events, and to explore the potential clinical benefit of enhanced IL-33/ST2 signalling in atherosclerosis.
Keywords: ST2, interleukin-33, atherosclerosis, inflammation, plaque
Abbreviations: ACS, acute coronary syndrome; CAD, coronary artery disease; CANTOS, Canakinumab Antiinflammatory Thrombosis Outcome Study; HF, heart failure; IL, Interleukin; IL2
ACCEPTED MANUSCRIPT 1RAcP, IL-1R accessory protein; MI, myocardial infarction; ST, suppression of tumorigenesis (ST2L, ST2-ligand; sST2, soluble ST2); Th, T helper. Inflammation and atherosclerosis: an overview The inflammatory hypothesis of atherosclerotic disease was formulated based on the presence of inflammatory cells in coronary plaques from patients with unstable angina,[1,2,3,4] along with the
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finding of a prognostic value of C-reactive protein for acute coronary syndromes (ACS),[5] and the
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evidence of neutrophil activation across the coronary vascular bed in patients with unstable angina.[6] Over time, an extensive body of evidence has supported this hypothesis, suggesting the
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beneficial effects of countering inflammation to delay atherosclerosis progression, and then improve
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prognosis of patients with coronary artery disease (CAD).[7] Although chronic inflammation by itself may not be causative of atherosclerosis,[8] it is believed to act synergistically with other risk
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factors for CAD, enhancing plaque formation and destabilization. Despite these premises, no strictly anti-inflammatory drugs have been evaluated in clinical trials until very recently.[9]
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The recent Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) trial has
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been hailed as the first demonstration of the inflammatory hypothesis of atherotrombosis,[10] and has boosted research on inflammation in CAD. Canakinumab is a monoclonal antibody targeting
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interleukin-1β (IL-1β), which is a potent pro-inflammatory cytokine that is crucial for host-defence responses to infection and injury. Canakinumab, administered at a dose of 150 mg every 3 months,
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caused a significant reduction in cardiovascular events compared to placebo in patients with previous MI, despite no lipid-level lowering effects.[10] In particular, the primary endpoint was a composite of nonfatal myocardial infarction (MI), nonfatal stroke, or cardiovascular death, while the secondary endpoint also included hospitalization for unstable angina requiring urgent revascularization.[10] Therefore, this study introduced IL-1β inhibition as a possible additional approach to secondary prevention after acute MI. On the other hand, concerns about the drug safety profile exist, mainly because of the higher rate of deaths for infection or sepsis in patients on
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ACCEPTED MANUSCRIPT canakinumab, possibly because canakinumab blocks a crucial mechanism of the immune response to pathogens.[9,10]
The IL-33/ST2 pathway: conceptual framework for its involvement in atherosclerosis Interleukin (IL)-33 is the most recently discovered member of the IL-1 cytokine family. Contrary to
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other members of the family, IL-33 bears a nuclear localization signal and a chromatin-binding
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motif, but lacks a secretion signalling sequence.[11] Thus, IL-33 is mainly released by damaged or
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necrotic barrier cells (endothelial and epithelial cells), acting as an alarmin, which is an endogenous factor eliciting proinflammatory responses.[11] Acting on several cell types and interacting with
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other cytokines, IL-33 can also modulate both innate and adaptive immune responses. IL-33 is expressed in almost all organs. Although IL-33 may act directly as a transcriptional regulator, its
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effects are mostly mediated by binding to a membrane receptor named suppression of tumorigenesis 2-ligand (ST2L).[11] IL-33 binding to ST2L leads to IL-1R accessory protein (IL-1RAcP)
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recruitment and the formation of a heterodimeric signalling complex that involves myeloid
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differentiation primary response protein 88 (MYD88), IL-1R-associated kinase 1 (IRAK1), IRAK4 and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and the activation of
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mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB).[11] Alternative promoter splicing and 3’ processing of the same mRNA of ST2L produces a decoy
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receptor named soluble ST2 (sST2).[12] sST2 is released into the blood stream and avidly binds IL33, preventing its interaction with ST2L/IL-1RAcP, and thus blocking its effects on target cells.[12] Since IL-33, ST2L and sST2 are all upregulated during cell stress, sST2 can be interpreted as a regulatory mechanism avoiding excessive IL-33 stimulation.[12] sST2 expression is almost ubiquitous in the body, and occurs mostly in endothelial cells in response to tissue damage and inflammation.[11,12] ST2L is expressed on different cell types including T cells. T cells include the T helper (Th) subset, which in turn is divided into two main subgroups: Th1 cells, which evoke cell-mediated 4
ACCEPTED MANUSCRIPT immunity and phagocyte-dependent inflammation, and Th2 cells, which elicit strong antibody responses and eosinophil accumulation, but inhibit several functions of phagocytic cells. A dynamic balance between Th1 and Th2 responses exists, and is influenced by Th1 and Th2 cytokines.[13] Excessive Th1 activation underlies several disorders characterized by chronic inflammation, including atherosclerosis, whereas a sustained Th2 response has been related mainly to allergic
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manifestations.[13]
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IL-33 has been characterized as an inducer of Th2 immune responses that does not cause a
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profound depression of Th1 responses.[14] Indeed, IL-33 promotes differentiation of naïve T cells into Th2 cells and activates resting Th2 cells; the mechanisms are either IL-33 binding to ST2L on
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T cells or enhanced production of Th2 cytokines in innate lymphoid cells.[14] A correlate of Th2 activation is blunting of Th1 immunity, as demonstrated by the fact that IL-33 relieves Th1-
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mediated disorders such as experimental arthritis [15] or autoimmune encephalitis.[16] It has also been reported that IL-33 can influence the phenotype and functions of macrophages. These cells,
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responding to environmental stimuli, can differentiate into M1 macrophages, inflammatory cells
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producing proinflammatory cytokines, or M2 macrophages, involved in tissue repair and resolution of inflammation.[17] IL-33 cooperates with other cytokines in eliciting macrophage transition to the
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M2 phenotype,[14] which has been associated with plaque fibrosis and greater stability.[18] Serum sST2 levels are often increased in inflammatory conditions and are considered a powerful
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predictor of prognosis in heart failure (HF). Indeed, higher sST2 levels have been consistently associated with a greater risk of all-cause and cardiovascular death in both chronic [19] and acute settings,[20] and with hospitalization after acute HF.[20] Circulating sST2 is also an independent predictor of adverse left ventricular remodelling and death after MI.[ 21 ] The prognostic significance of sST2 may be explained by its deleterious effects on the heart, where it abolishes the cardioprotective effects of the IL-33/ST2 interaction, as well as its correlation with the intensity of the systemic inflammatory and profibrotic response.[12,19,20]
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ACCEPTED MANUSCRIPT Another possible explanation of the link between sST2 and prognosis is its association with the severity of atherosclerotic disease and the propensity to plaque destabilization. In fact, IL-33 and both the cell-bound and soluble forms of ST2 are expressed in human coronary and carotid plaques,[22,23] and it may be speculated that IL-33 inhibits the immune response involved in atherosclerosis (both Th1 and M1), while sST2 promotes it.
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The relationship between the IL-33/ST2 pathway and atherosclerosis has been explored at
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several levels. First, animal models of accelerated atherosclerosis have been treated with IL-33,
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alone or together with sST2, in order to explore the effects on plaque extent and inflammation. Second, sST2 levels, or gene polymorphisms influencing the IL-33/ST2 pathway, have been
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correlated with atherosclerotic burden, plaque inflammation and prognosis in patients.
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Animal studies
In ApoE-/- mice on a high-fat diet, IL-33 administration over 6 weeks was associated with reduced
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plaque size in the aortic sinus.[24] This finding was ascribed to reduced in vitro production of foam
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cells from macrophages. The proposed mechanisms were: 1) a polarization of the immune response towards the Th2 phenotype, 2) the inhibition of foam cell formation from macrophages, 3) the
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induction of antibodies targeting oxidized low density lipoproteins (ox-LDL).[24] Indeed, mice receiving IL-33 had higher levels of Th2 cytokines (such as IL-5) in serum and lymph node cells, as
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well as antibodies targeting ox-LDL, whose accumulation in macrophages would produce foam cells. IL-5 seemed to be an important mediator of IL-33-induced antibody response against oxLDL,[24] in agreement with a previous report.[25] A reduced formation of foam cells following the IL-33/ST2L interaction has been also confirmed in another study.[26] In the same model, IL-33 administration reduced plaque inflammation, as denoted by a lower degree of macrophage and T-cell accumulation,[24] possibly because of a reduced foam cell content and a shift towards a Th2, M2 immune response. Finally, concomitant IL-33 and sST2
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ACCEPTED MANUSCRIPT administration was associated with a greater atherosclerosis burden and an enhanced Th1 response.[24] To further explore the function of the IL-33/ST2 pathway, its silencing was pursued. ApoE-/-, ApoE-/- IL-33-/-, ApoE-/- ST2-/- or ApoE-/- mice injected with a neutralizing anti-ST2 antibody were compared. The lipid-staining area in the thoracic-abdominal aorta and the aortic sinus, along
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with the Th1/Th2 cytokine response of lymph node cells to in vitro stimulation did not differ
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significantly.[27] The diverging conclusions of these two studies may be explained by technical
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considerations, such as heterogeneity in diet and methods to calculate plaque extent.[27] Furthermore, the effect of IL-33/ST2 modulation was assessed mainly in terms of atherosclerotic
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burden, although plaque inflammation shows a closer association with acute plaque changes than plaque growth and final size. Further considerations on the animal models are provided in the
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concluding remarks.
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Human studies
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Several studies have tried to correlate gene variants in the IL33/ST2L pathway with the risk of CAD. When assessing 1146 CAD patients and age- and sex-matched controls, no significant
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association between gene polymorphisms in the IL-33 or ST2 genes and presence/absence of CAD was demonstrated.[28] Similar results were found when comparing patients with previous MI vs.
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control subjects.[29] Notably, the relevance of these analyses is limited by the small number of gene variants explored, and by the lack of genotype-phenotype correlations, making it difficult to clarify the relationship with ST2/IL-33 signalling. On the other hand, a case-control analysis identified the association of rs7025417 genotype with higher circulating IL-33, and with lower risk for CAD (defined as >70% luminal stenosis in at least one main vessel at coronary angiography, previous coronary artery bypass graft, percutaneous coronary intervention, and/or MI).[30] In line with the hypothesis of a protective role of IL-33, some clues of a negative prognostic role of higher circulating sST2 exist. In 456 individuals undergoing cardiac computed tomography, 7
ACCEPTED MANUSCRIPT serum sST2 level displayed a significant association with a surrogate index of atherosclerotic burden, i.e. a coronary artery calcium score >300 Agatston unit, although sST2 resulted less predictive than C-reactive protein.[ 31 ] On the other hand, sST2 did not correlate with the angiographic severity of CAD in a single centre study evaluating 1345 patients with stable CAD referred for invasive coronary evaluation. With regard to outcome prediction, sST2 was an
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independent predictor of both all-cause and cardiovascular mortality in a model including high-
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sensitivity cardiac troponin T and N-terminal fraction of pro-B-type natriuretic peptide, but
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endpoints more closely related with plaque destabilization, such as MI occurrence, were not evaluated.[32]
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Few studies have investigated the IL-33/ST2 pathway in carotid plaques of patients undergoing endarterectomy. In a cohort of 391 patients, sST2 did not correlate with the histological features of
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the carotid plaque nor with cardiovascular events along a 3-year follow-up.[33] Another study demonstrated a higher expression of ST2L on macrophages within carotid artery plaques from
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patients with cerebrovascular symptoms than in those without.[34] The authors interpreted these
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findings by postulating a pro-inflammatory effect of the IL-33/ST2L signalling,[34] citing additional data that point to a role for IL-33 in endothelial activation, leukocyte migration, and
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angiogenesis.[35,36] This conclusion is in disagreement with our current understanding of this pathway, and enhanced ST2L expression may as well represent a negative feedback mechanism
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aiming to limit plaque inflammation. Nonetheless, as in the case for animal studies, no clear message can be derived from human studies, and there is a clear need for further evaluation of the link between the IL-33/ST2 axis and plaque inflammation.
The IL-33/ST2 axis in atherosclerosis: where do we stand? The recent CANTOS trial disclosed the attractive opportunity of improving the prognosis of atherosclerotic disease by modulating inflammatory pathways.[10] IL-1β can be produced by both intrinsic vascular wall cells and lesional leukocytes, and this cytokine may act on endothelial cells, 8
ACCEPTED MANUSCRIPT smooth muscle cells and leukocytes promoting the development of atherosclerotic plaques, as reviewed very recently.[37] Nevertheless, IL-1β plays a central role also in the regulation of the immune response to infections, possibly explaining the greater propensity to infection and sepsis among patients on canakinumab in the CANTOS trial.[10] Although this effect was counterbalanced by a reduction in cancer-related mortality,[10] as confirmed in a dedicated study
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on lung cancer,[38] dissecting the mechanisms of plaque inflammation may allow to identify more
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specific targets for treatment. Furthermore, circulating biomarkers of plaque inflammation may
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prove indicators of the severity of atherosclerotic burden and/or the likelihood of acute plaque changes.
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Despite being far from specific for plaque inflammation (and having no direct link with the IL1β signalling blocked by canakinumab),[14] the IL-33/ST2 pathway has several elements of
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interest. It may be easily modulated through IL-33 or IL-33-targeting antibodies, and activating the IL-33/ST2L signalling should slow down the development of atherosclerosis by blunting the Th1
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and M1 immune response. Furthermore, the IL-33/ST2L interaction is cardioprotective, and the
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safety profile of a polarization of immune responses towards a Th2, M2 phenotype may be more acceptable than the blockade of upstream regulators of inflammatory responses (such as IL-1β). In
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summary, the IL-33/ST2 interaction would slow down plaque evolution, while sST2 would have deleterious effects by sequestering IL-33 (Figure 1). Following IL-33/ST2L binding on the surface
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of immune cells, differentiation towards a Th2, M2 phenotype would ensue. Th2 cells would inhibit phagocytosis and trigger an antibody response targeting also ox-LDL. These mechanisms, together with macrophage differentiation into M2 cells, would limit foam cell production (Figure 2). Though plausible, all these considerations remain largely speculative. Despite a solid theoretical framework, no definite demonstration of an involvement of the IL-33/ST2 pathway in atherosclerosis has been provided. Moreover, currently available evidence does not allow to draw any conclusions about a relationship between IL-33/ST2 activation and atherosclerotic burden, on the one hand, and plaque vulnerability, on the other hand. 9
ACCEPTED MANUSCRIPT Several approaches may provide the missing evidence needed to clearly demonstrate the involvement of the IL-33/ST2 pathway in atherosclerosis. Characterization of plaques from either animal models or human patients remains a promising strategy, despite several limitations. For example, animal models with abnormally high cholesterol levels and profound alterations of the IL33/ST2 pathways (such as gene knockout) may fail to reproduce closely the human phenotype. In
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humans, specimens from carotid endarterectomy are those most commonly available, although the
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mechanisms of atherosclerosis onset and progression are not necessarily the same in the two
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districts; for this reason, the assessment of coronary plaques from endarterectomy, post-mortem examinations, or explanted hearts is worth considering. Finally, it could prove hard to define the
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role of the IL-33/ST2 pathway based on the expression of its constituents within the plaque. Therefore, histological studies should be carried out and interpreted together with biohumoral
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analyses, aiming to characterize circulating IL-33 and sST2 as indicators of atherosclerotic burden and/or predictors of ACS. Finally, a cause-effect relationship between gene variants impacting on
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the IL-33/ST2 pathway (for example, increasing IL-33 expression), and the development of
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atherosclerosis and its complications may be searched through genome-wide association studies or a Mendelian randomization approach.[39]
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In conclusions, IL-33 and sST2 might be evaluated as indicators of the atherosclerotic burden, and mostly as predictors of future coronary and/or cerebrovascular events. In particular, sST2 is
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emerging as a powerful prognostic indicator in HF [40,41] and following MI,[42] and there is a theoretical framework for it being a promoter of plaque destabilization. Future studies should determine if sST2 may help predict cardiovascular events in the general population and/or in patients with known atherosclerotic disease.
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ACCEPTED MANUSCRIPT Figure legends Figure 1. Proposed role of the IL-33/ST2 pathway in atherosclerosis. In the arterial wall, the interaction between interleukin-33 (IL-33) and a membrane-bound receptor named suppression of tumorigenesis-2 ligand (ST2L) directs the immune response towards a T helper 2 (Th2), macrophage 2 (M2) phenotype; this limits plaque inflammation and evolution. The
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circulating isoform of ST2L, named soluble ST2 (sST2), blocks the protective effects of IL-33 on
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atherosclerotic plaques by sequestering IL-33.
Figure 2. Possible roles of the IL-33/ST2 pathway in the determinism of plaque inflammation.
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The interaction between interleukin-33 (IL-33) and suppression of tumorigenesis-2 ligand (ST2L) on the surface of immune cells (either T helper - Th - or macrophages) promotes differentiation
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towards a Th2 or M2 phenotype, respectively. Soluble ST2 (sST2) acts as a decoy receptor for IL33, blocking its effects on target cells. The Th2 cells inhibit macrophage phagocytosis and reduce
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plaque inflammation, as well as releasing cytokines that sustain further production of Th2 cells. In
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parallel, macrophage differentiation shift towards the M2 phenotype; this mechanism limits the
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formation of foam cells, and then lipid accumulation within the plaque.
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[23] Demyanets S, Kaun C, Pentz R, et al. Components of the interleukin-33/ST2 system are differentially expressed and regulated in human cardiac cells and in cells of the cardiac vasculature. J Mol Cell Cardiol 2013;60:16-26. [24] Miller AM, Xu D, Asquith DL, et al IL-33 reduces the development of atherosclerosis. J Exp
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[33] Willems S, Quax PH, de Borst GJ, et al. Soluble ST2 levels are not associated with secondary cardiovascular events and vulnerable plaque phenotype in patients with carotid artery stenosis. Atherosclerosis 2013;231:48-53. [34] Marzullo A, Ambrosi F, Inchingolo M, et al. ST2L transmembrane receptor expression: an
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[ 36 ] Demyanets S, Konya V, Kastl SP, et al. Interleukin-33 induces expression of adhesion molecules and inflammatory activation in human endothelial cells and in human atherosclerotic
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plaques. Arterioscler Thromb Vasc Biol 2011; 31:2080–2089.
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canakinumab on incident lung cancer in patients with atherosclerosis: exploratory results from a randomized, double-blind, placebo-controlled trial. Lancet. 2017;390:1833-1842. [39] Ebrahim S, Davey Smith G. Mendelian randomization: can genetic epidemiology help redress
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[40] Maisel AS, Richards AM, Pascual-Figal D, Mueller C. Serial ST2 testing in hospitalized patients with acute heart failure. Am J Cardiol 2015;115:32B-37B. [41] Bayes-Genis A, Zhang Y, Ky B. ST2 and patient prognosis in chronic heart failure. Am J Cardiol 2015;115:64B-69B. [42] Richards AM, Di Somma S, Mueller T. ST2 in stable and unstable ischemic heart diseases. Am J Cardiol 2015;115:48B-58B.
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Highlights - Knowledge on the IL-33/ST2 pathway in atherosclerosis is limited. - The elements of the IL-33/ST2 pathway are expressed in the endothelium of arterial vessels. - IL-33 blunts the immune response characteristic of atherosclerosis.
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- Soluble ST2 acts as a decoy receptor for IL-33, thus blocking its protective effects.
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Figure 1
Figure 2
Alberto Aimo, Paola Migliorini, Giuseppe Vergaro, Maria Franzini, Claudio Passino, Alan Maisel, Michele Emdin PII: DOI: Reference:
S0167-5273(18)30957-4 doi:10.1016/j.ijcard.2018.05.056 IJCA 26469
To appear in: Received date: Revised date: Accepted date:
8 February 2018 19 April 2018 17 May 2018
Please cite this article as: Alberto Aimo, Paola Migliorini, Giuseppe Vergaro, Maria Franzini, Claudio Passino, Alan Maisel, Michele Emdin , The IL-33/ST2 pathway, inflammation and atherosclerosis: Trigger and target?. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ijca(2017), doi:10.1016/j.ijcard.2018.05.056
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ACCEPTED MANUSCRIPT The IL-33/ST2 Pathway, Inflammation and Atherosclerosis: Trigger and Target?
Alberto Aimo, MD,1,2 Paola Migliorini, MD, PhD,3 Giuseppe Vergaro, MD, PhD,1,4 Maria Franzini,
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PhD,5 Claudio Passino, MD,1,4 Alan Maisel, MD,6 Michele Emdin, MD, PhD1,4
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Author affiliations:
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2. Cardiology Division, University of Pisa, Italy
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1. Institute of Life Sciences, Scuola Superiore Sant’Anna, Pisa, Italy
3. Allergy and Immunology Division, University of Pisa, Pisa, Italy
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4. Cardiology Division, Fondazione Toscana Gabriele Monasterio, Pisa, Italy 5. Clinical Pathology Unit, University of Pisa, Italy
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6. University of California, San Diego, CA, USA.
Conflicts of interest: A.M. is consultant for Critical Diagnostics
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Word counts: 2511 (text)
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Address for correspondence: Alberto Aimo, MD
Scuola Superiore Sant'Anna and Cardiology Division, University of Pisa Piazza Martiri della Libertà, 33, Pisa, Italy Tel +39 050883111 Fax +39 050883225 Phone +39 3477084391 E-mail: [email protected], [email protected] 1
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Abstract The “inflammatory hypothesis” of atherosclerosis postulates that inflammatory cell signalling drives the formation, growth and ultimately the instability of atherosclerotic plaques, setting up the
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substrate for the thrombotic response that causes myocardial damage or infarction. The recent
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Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) trial has been hailed as
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the first demonstration, ex iuvantibus, of the inflammatory hypothesis. Indeed, interleukin (IL)-1β inhibition was found to reduce cardiovascular events in patients with previous myocardial infarction
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and raised high-sensitivity C-reactive protein, despite no effects on the lipid profile. These results prompt a dissection of inflammatory mechanisms of atherosclerosis in order to search for specific
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biomarkers with prognostic value and/or therapeutic targets.
Under this respect, the IL-33/suppression of tumorigenesis 2 (ST2) pathway deserves
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consideration. Indeed, its elements are particularly expressed in the endothelium of arterial vessels,
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and the interaction between IL-33 and the ST2 receptor blunts the immune response characteristic of atherosclerosis. By contrast, soluble ST2 (sST2) acts as a decoy receptor for IL-33, thus blocking
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its protective effects. Despite a solid theoretical framework, no definite demonstration of an involvement of the IL-33/ST2 pathway in atherosclerosis has been provided. Therefore, further
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studies are warranted to verify if elements of the IL-33/ST2 pathway may be proposed as markers of plaque burden and predictors of future cardiovascular events, and to explore the potential clinical benefit of enhanced IL-33/ST2 signalling in atherosclerosis.
Keywords: ST2, interleukin-33, atherosclerosis, inflammation, plaque
Abbreviations: ACS, acute coronary syndrome; CAD, coronary artery disease; CANTOS, Canakinumab Antiinflammatory Thrombosis Outcome Study; HF, heart failure; IL, Interleukin; IL2
ACCEPTED MANUSCRIPT 1RAcP, IL-1R accessory protein; MI, myocardial infarction; ST, suppression of tumorigenesis (ST2L, ST2-ligand; sST2, soluble ST2); Th, T helper. Inflammation and atherosclerosis: an overview The inflammatory hypothesis of atherosclerotic disease was formulated based on the presence of inflammatory cells in coronary plaques from patients with unstable angina,[1,2,3,4] along with the
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finding of a prognostic value of C-reactive protein for acute coronary syndromes (ACS),[5] and the
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evidence of neutrophil activation across the coronary vascular bed in patients with unstable angina.[6] Over time, an extensive body of evidence has supported this hypothesis, suggesting the
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beneficial effects of countering inflammation to delay atherosclerosis progression, and then improve
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prognosis of patients with coronary artery disease (CAD).[7] Although chronic inflammation by itself may not be causative of atherosclerosis,[8] it is believed to act synergistically with other risk
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factors for CAD, enhancing plaque formation and destabilization. Despite these premises, no strictly anti-inflammatory drugs have been evaluated in clinical trials until very recently.[9]
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The recent Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) trial has
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been hailed as the first demonstration of the inflammatory hypothesis of atherotrombosis,[10] and has boosted research on inflammation in CAD. Canakinumab is a monoclonal antibody targeting
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interleukin-1β (IL-1β), which is a potent pro-inflammatory cytokine that is crucial for host-defence responses to infection and injury. Canakinumab, administered at a dose of 150 mg every 3 months,
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caused a significant reduction in cardiovascular events compared to placebo in patients with previous MI, despite no lipid-level lowering effects.[10] In particular, the primary endpoint was a composite of nonfatal myocardial infarction (MI), nonfatal stroke, or cardiovascular death, while the secondary endpoint also included hospitalization for unstable angina requiring urgent revascularization.[10] Therefore, this study introduced IL-1β inhibition as a possible additional approach to secondary prevention after acute MI. On the other hand, concerns about the drug safety profile exist, mainly because of the higher rate of deaths for infection or sepsis in patients on
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ACCEPTED MANUSCRIPT canakinumab, possibly because canakinumab blocks a crucial mechanism of the immune response to pathogens.[9,10]
The IL-33/ST2 pathway: conceptual framework for its involvement in atherosclerosis Interleukin (IL)-33 is the most recently discovered member of the IL-1 cytokine family. Contrary to
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other members of the family, IL-33 bears a nuclear localization signal and a chromatin-binding
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motif, but lacks a secretion signalling sequence.[11] Thus, IL-33 is mainly released by damaged or
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necrotic barrier cells (endothelial and epithelial cells), acting as an alarmin, which is an endogenous factor eliciting proinflammatory responses.[11] Acting on several cell types and interacting with
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other cytokines, IL-33 can also modulate both innate and adaptive immune responses. IL-33 is expressed in almost all organs. Although IL-33 may act directly as a transcriptional regulator, its
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effects are mostly mediated by binding to a membrane receptor named suppression of tumorigenesis 2-ligand (ST2L).[11] IL-33 binding to ST2L leads to IL-1R accessory protein (IL-1RAcP)
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recruitment and the formation of a heterodimeric signalling complex that involves myeloid
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differentiation primary response protein 88 (MYD88), IL-1R-associated kinase 1 (IRAK1), IRAK4 and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and the activation of
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mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB).[11] Alternative promoter splicing and 3’ processing of the same mRNA of ST2L produces a decoy
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receptor named soluble ST2 (sST2).[12] sST2 is released into the blood stream and avidly binds IL33, preventing its interaction with ST2L/IL-1RAcP, and thus blocking its effects on target cells.[12] Since IL-33, ST2L and sST2 are all upregulated during cell stress, sST2 can be interpreted as a regulatory mechanism avoiding excessive IL-33 stimulation.[12] sST2 expression is almost ubiquitous in the body, and occurs mostly in endothelial cells in response to tissue damage and inflammation.[11,12] ST2L is expressed on different cell types including T cells. T cells include the T helper (Th) subset, which in turn is divided into two main subgroups: Th1 cells, which evoke cell-mediated 4
ACCEPTED MANUSCRIPT immunity and phagocyte-dependent inflammation, and Th2 cells, which elicit strong antibody responses and eosinophil accumulation, but inhibit several functions of phagocytic cells. A dynamic balance between Th1 and Th2 responses exists, and is influenced by Th1 and Th2 cytokines.[13] Excessive Th1 activation underlies several disorders characterized by chronic inflammation, including atherosclerosis, whereas a sustained Th2 response has been related mainly to allergic
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manifestations.[13]
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IL-33 has been characterized as an inducer of Th2 immune responses that does not cause a
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profound depression of Th1 responses.[14] Indeed, IL-33 promotes differentiation of naïve T cells into Th2 cells and activates resting Th2 cells; the mechanisms are either IL-33 binding to ST2L on
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T cells or enhanced production of Th2 cytokines in innate lymphoid cells.[14] A correlate of Th2 activation is blunting of Th1 immunity, as demonstrated by the fact that IL-33 relieves Th1-
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mediated disorders such as experimental arthritis [15] or autoimmune encephalitis.[16] It has also been reported that IL-33 can influence the phenotype and functions of macrophages. These cells,
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responding to environmental stimuli, can differentiate into M1 macrophages, inflammatory cells
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producing proinflammatory cytokines, or M2 macrophages, involved in tissue repair and resolution of inflammation.[17] IL-33 cooperates with other cytokines in eliciting macrophage transition to the
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M2 phenotype,[14] which has been associated with plaque fibrosis and greater stability.[18] Serum sST2 levels are often increased in inflammatory conditions and are considered a powerful
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predictor of prognosis in heart failure (HF). Indeed, higher sST2 levels have been consistently associated with a greater risk of all-cause and cardiovascular death in both chronic [19] and acute settings,[20] and with hospitalization after acute HF.[20] Circulating sST2 is also an independent predictor of adverse left ventricular remodelling and death after MI.[ 21 ] The prognostic significance of sST2 may be explained by its deleterious effects on the heart, where it abolishes the cardioprotective effects of the IL-33/ST2 interaction, as well as its correlation with the intensity of the systemic inflammatory and profibrotic response.[12,19,20]
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ACCEPTED MANUSCRIPT Another possible explanation of the link between sST2 and prognosis is its association with the severity of atherosclerotic disease and the propensity to plaque destabilization. In fact, IL-33 and both the cell-bound and soluble forms of ST2 are expressed in human coronary and carotid plaques,[22,23] and it may be speculated that IL-33 inhibits the immune response involved in atherosclerosis (both Th1 and M1), while sST2 promotes it.
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The relationship between the IL-33/ST2 pathway and atherosclerosis has been explored at
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several levels. First, animal models of accelerated atherosclerosis have been treated with IL-33,
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alone or together with sST2, in order to explore the effects on plaque extent and inflammation. Second, sST2 levels, or gene polymorphisms influencing the IL-33/ST2 pathway, have been
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correlated with atherosclerotic burden, plaque inflammation and prognosis in patients.
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Animal studies
In ApoE-/- mice on a high-fat diet, IL-33 administration over 6 weeks was associated with reduced
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plaque size in the aortic sinus.[24] This finding was ascribed to reduced in vitro production of foam
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cells from macrophages. The proposed mechanisms were: 1) a polarization of the immune response towards the Th2 phenotype, 2) the inhibition of foam cell formation from macrophages, 3) the
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induction of antibodies targeting oxidized low density lipoproteins (ox-LDL).[24] Indeed, mice receiving IL-33 had higher levels of Th2 cytokines (such as IL-5) in serum and lymph node cells, as
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well as antibodies targeting ox-LDL, whose accumulation in macrophages would produce foam cells. IL-5 seemed to be an important mediator of IL-33-induced antibody response against oxLDL,[24] in agreement with a previous report.[25] A reduced formation of foam cells following the IL-33/ST2L interaction has been also confirmed in another study.[26] In the same model, IL-33 administration reduced plaque inflammation, as denoted by a lower degree of macrophage and T-cell accumulation,[24] possibly because of a reduced foam cell content and a shift towards a Th2, M2 immune response. Finally, concomitant IL-33 and sST2
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ACCEPTED MANUSCRIPT administration was associated with a greater atherosclerosis burden and an enhanced Th1 response.[24] To further explore the function of the IL-33/ST2 pathway, its silencing was pursued. ApoE-/-, ApoE-/- IL-33-/-, ApoE-/- ST2-/- or ApoE-/- mice injected with a neutralizing anti-ST2 antibody were compared. The lipid-staining area in the thoracic-abdominal aorta and the aortic sinus, along
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with the Th1/Th2 cytokine response of lymph node cells to in vitro stimulation did not differ
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significantly.[27] The diverging conclusions of these two studies may be explained by technical
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considerations, such as heterogeneity in diet and methods to calculate plaque extent.[27] Furthermore, the effect of IL-33/ST2 modulation was assessed mainly in terms of atherosclerotic
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burden, although plaque inflammation shows a closer association with acute plaque changes than plaque growth and final size. Further considerations on the animal models are provided in the
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concluding remarks.
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Human studies
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Several studies have tried to correlate gene variants in the IL33/ST2L pathway with the risk of CAD. When assessing 1146 CAD patients and age- and sex-matched controls, no significant
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association between gene polymorphisms in the IL-33 or ST2 genes and presence/absence of CAD was demonstrated.[28] Similar results were found when comparing patients with previous MI vs.
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control subjects.[29] Notably, the relevance of these analyses is limited by the small number of gene variants explored, and by the lack of genotype-phenotype correlations, making it difficult to clarify the relationship with ST2/IL-33 signalling. On the other hand, a case-control analysis identified the association of rs7025417 genotype with higher circulating IL-33, and with lower risk for CAD (defined as >70% luminal stenosis in at least one main vessel at coronary angiography, previous coronary artery bypass graft, percutaneous coronary intervention, and/or MI).[30] In line with the hypothesis of a protective role of IL-33, some clues of a negative prognostic role of higher circulating sST2 exist. In 456 individuals undergoing cardiac computed tomography, 7
ACCEPTED MANUSCRIPT serum sST2 level displayed a significant association with a surrogate index of atherosclerotic burden, i.e. a coronary artery calcium score >300 Agatston unit, although sST2 resulted less predictive than C-reactive protein.[ 31 ] On the other hand, sST2 did not correlate with the angiographic severity of CAD in a single centre study evaluating 1345 patients with stable CAD referred for invasive coronary evaluation. With regard to outcome prediction, sST2 was an
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independent predictor of both all-cause and cardiovascular mortality in a model including high-
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sensitivity cardiac troponin T and N-terminal fraction of pro-B-type natriuretic peptide, but
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endpoints more closely related with plaque destabilization, such as MI occurrence, were not evaluated.[32]
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Few studies have investigated the IL-33/ST2 pathway in carotid plaques of patients undergoing endarterectomy. In a cohort of 391 patients, sST2 did not correlate with the histological features of
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the carotid plaque nor with cardiovascular events along a 3-year follow-up.[33] Another study demonstrated a higher expression of ST2L on macrophages within carotid artery plaques from
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patients with cerebrovascular symptoms than in those without.[34] The authors interpreted these
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findings by postulating a pro-inflammatory effect of the IL-33/ST2L signalling,[34] citing additional data that point to a role for IL-33 in endothelial activation, leukocyte migration, and
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angiogenesis.[35,36] This conclusion is in disagreement with our current understanding of this pathway, and enhanced ST2L expression may as well represent a negative feedback mechanism
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aiming to limit plaque inflammation. Nonetheless, as in the case for animal studies, no clear message can be derived from human studies, and there is a clear need for further evaluation of the link between the IL-33/ST2 axis and plaque inflammation.
The IL-33/ST2 axis in atherosclerosis: where do we stand? The recent CANTOS trial disclosed the attractive opportunity of improving the prognosis of atherosclerotic disease by modulating inflammatory pathways.[10] IL-1β can be produced by both intrinsic vascular wall cells and lesional leukocytes, and this cytokine may act on endothelial cells, 8
ACCEPTED MANUSCRIPT smooth muscle cells and leukocytes promoting the development of atherosclerotic plaques, as reviewed very recently.[37] Nevertheless, IL-1β plays a central role also in the regulation of the immune response to infections, possibly explaining the greater propensity to infection and sepsis among patients on canakinumab in the CANTOS trial.[10] Although this effect was counterbalanced by a reduction in cancer-related mortality,[10] as confirmed in a dedicated study
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on lung cancer,[38] dissecting the mechanisms of plaque inflammation may allow to identify more
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specific targets for treatment. Furthermore, circulating biomarkers of plaque inflammation may
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prove indicators of the severity of atherosclerotic burden and/or the likelihood of acute plaque changes.
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Despite being far from specific for plaque inflammation (and having no direct link with the IL1β signalling blocked by canakinumab),[14] the IL-33/ST2 pathway has several elements of
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interest. It may be easily modulated through IL-33 or IL-33-targeting antibodies, and activating the IL-33/ST2L signalling should slow down the development of atherosclerosis by blunting the Th1
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and M1 immune response. Furthermore, the IL-33/ST2L interaction is cardioprotective, and the
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safety profile of a polarization of immune responses towards a Th2, M2 phenotype may be more acceptable than the blockade of upstream regulators of inflammatory responses (such as IL-1β). In
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summary, the IL-33/ST2 interaction would slow down plaque evolution, while sST2 would have deleterious effects by sequestering IL-33 (Figure 1). Following IL-33/ST2L binding on the surface
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of immune cells, differentiation towards a Th2, M2 phenotype would ensue. Th2 cells would inhibit phagocytosis and trigger an antibody response targeting also ox-LDL. These mechanisms, together with macrophage differentiation into M2 cells, would limit foam cell production (Figure 2). Though plausible, all these considerations remain largely speculative. Despite a solid theoretical framework, no definite demonstration of an involvement of the IL-33/ST2 pathway in atherosclerosis has been provided. Moreover, currently available evidence does not allow to draw any conclusions about a relationship between IL-33/ST2 activation and atherosclerotic burden, on the one hand, and plaque vulnerability, on the other hand. 9
ACCEPTED MANUSCRIPT Several approaches may provide the missing evidence needed to clearly demonstrate the involvement of the IL-33/ST2 pathway in atherosclerosis. Characterization of plaques from either animal models or human patients remains a promising strategy, despite several limitations. For example, animal models with abnormally high cholesterol levels and profound alterations of the IL33/ST2 pathways (such as gene knockout) may fail to reproduce closely the human phenotype. In
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humans, specimens from carotid endarterectomy are those most commonly available, although the
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mechanisms of atherosclerosis onset and progression are not necessarily the same in the two
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districts; for this reason, the assessment of coronary plaques from endarterectomy, post-mortem examinations, or explanted hearts is worth considering. Finally, it could prove hard to define the
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role of the IL-33/ST2 pathway based on the expression of its constituents within the plaque. Therefore, histological studies should be carried out and interpreted together with biohumoral
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analyses, aiming to characterize circulating IL-33 and sST2 as indicators of atherosclerotic burden and/or predictors of ACS. Finally, a cause-effect relationship between gene variants impacting on
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the IL-33/ST2 pathway (for example, increasing IL-33 expression), and the development of
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atherosclerosis and its complications may be searched through genome-wide association studies or a Mendelian randomization approach.[39]
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In conclusions, IL-33 and sST2 might be evaluated as indicators of the atherosclerotic burden, and mostly as predictors of future coronary and/or cerebrovascular events. In particular, sST2 is
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emerging as a powerful prognostic indicator in HF [40,41] and following MI,[42] and there is a theoretical framework for it being a promoter of plaque destabilization. Future studies should determine if sST2 may help predict cardiovascular events in the general population and/or in patients with known atherosclerotic disease.
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ACCEPTED MANUSCRIPT Figure legends Figure 1. Proposed role of the IL-33/ST2 pathway in atherosclerosis. In the arterial wall, the interaction between interleukin-33 (IL-33) and a membrane-bound receptor named suppression of tumorigenesis-2 ligand (ST2L) directs the immune response towards a T helper 2 (Th2), macrophage 2 (M2) phenotype; this limits plaque inflammation and evolution. The
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circulating isoform of ST2L, named soluble ST2 (sST2), blocks the protective effects of IL-33 on
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atherosclerotic plaques by sequestering IL-33.
Figure 2. Possible roles of the IL-33/ST2 pathway in the determinism of plaque inflammation.
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The interaction between interleukin-33 (IL-33) and suppression of tumorigenesis-2 ligand (ST2L) on the surface of immune cells (either T helper - Th - or macrophages) promotes differentiation
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towards a Th2 or M2 phenotype, respectively. Soluble ST2 (sST2) acts as a decoy receptor for IL33, blocking its effects on target cells. The Th2 cells inhibit macrophage phagocytosis and reduce
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plaque inflammation, as well as releasing cytokines that sustain further production of Th2 cells. In
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parallel, macrophage differentiation shift towards the M2 phenotype; this mechanism limits the
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formation of foam cells, and then lipid accumulation within the plaque.
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[33] Willems S, Quax PH, de Borst GJ, et al. Soluble ST2 levels are not associated with secondary cardiovascular events and vulnerable plaque phenotype in patients with carotid artery stenosis. Atherosclerosis 2013;231:48-53. [34] Marzullo A, Ambrosi F, Inchingolo M, et al. ST2L transmembrane receptor expression: an
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Highlights - Knowledge on the IL-33/ST2 pathway in atherosclerosis is limited. - The elements of the IL-33/ST2 pathway are expressed in the endothelium of arterial vessels. - IL-33 blunts the immune response characteristic of atherosclerosis.
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- Soluble ST2 acts as a decoy receptor for IL-33, thus blocking its protective effects.
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Figure 1
Figure 2