Curr Heart Fail Rep DOI 10.1007/s11897-015-0255-7

COMORBIDITIES OF HEART FAILURE (CE ANGERMANN, SECTION EDITOR)

Cellular Immunity and Cardiac Remodeling After Myocardial Infarction: Role of Neutrophils, Monocytes, and Macrophages Hisahito Shinagawa & Stefan Frantz

# Springer Science+Business Media New York 2015

Abstract Today, innate immunity is recognized as an important pathophysiologic factor and therapeutic target for cardiac remodeling after myocardial infarction (MI). The innate immune system exerts its function via soluble and cellular components. Recently, function and kinetics of immune cells after MI have been clarified using new innovative technology. Therefore, herein, we will discuss the function of neutrophils, monocytes, and macrophages in the pathophysiology of cardiac remodeling after MI in basic as well as clinical science. Keywords Neutrophil . Monocyte . Macrophage . Innate immunity . Heart failure . Cardiac remodeling . Myocardial infarction

Introduction Current heart failure (HF) treatment targets mainly neurohumoral factors. β-Blocker, mineralocorticoid receptor antagonists, and angiotensin-converting enzyme inhibitor (ACEI) This article is part of the Topical Collection on Comorbidities of Heart Failure H. Shinagawa Department of Cardiovascular Medicine, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan H. Shinagawa : S. Frantz Comprehensive Heart Failure Center, University of Würzburg, Würzburg, Germany S. Frantz (*) Universitätsklinik und Poliklinik für Innere Medizin III, Universitätsklinikum Halle (Saale), Ernst-Grube-Straße 40, 06120 Halle (Saale), Germany e-mail: [email protected]

are the cornerstones of current therapeutic algorithms; however, we lack drugs with innovative targets based on new pathophysiologic concepts to date. Although there are no animal models which are strictly comparable with the complex and heterogeneous clinical picture of heart failure, the experimental coronary artery ligation model may share some pathomechanisms and correlate with remodeling after myocardial infarction. Indeed, in basic science, it has been very well shown that activation of the innate immune system contributes to heart failure development. Immunomodulation was, therefore, tried as an option for heart failure treatment. However, several clinical trials with immunomodulating/ antiinflammatory targets had disappointing results [1]. For example, heart failure patients treated with a tumor necrosis factor alpha (TNF-α) antagonist had no clinical improvement and even increased risk of hospitalization and death in the highdose group [2]. Indeed, depending on the circumstances, activation of the immune system can be protective or detrimental. Therefore, the negative results are most likely due to a lack of understanding by what, how, and in what time frame the immune system is activated. Recent progress in imaging tools and molecular techniques made it possible to describe precise movement and function of immune cells at different time points. Especially, investigations of monocyte/macrophage kinetics in the heart [3••] are remarkable since they are essential for healing after MI and are not built within the heart, but in the spleen/bone marrow. A better understanding and imaging of immune cells might therefore help to develop and guide anti-inflammatory therapeutic approaches and will be the focus of the current review.

Neutrophils After Myocardial Infarction The role of neutrophils in the field of HF has been extensively analyzed focusing on post-infarct inflammation and

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remodeling [4, 5]. Neutrophils are the first cells massively invading the myocardium after myocardial infarction (MI). They play a pivotal role in intensifying inflammation and initiating the cascade of wound healing after MI. Peripheral neutrophil count and neutrophil/lymphocyte ratio (NLR) predict adverse prognosis [6] and remodeling [4] after MI. Even after percutaneous coronary intervention in ST elevation MI, increased neutrophil counts are prognostic for larger infarct size and adverse cardiac function [4]. Antibodymediated depletion of neutrophils in a canine ischemic reperfusion (I/R) model reduced infarct size [7]. However, when increasing ischemic time, the depletion of neutrophil had less of a protective effect. This indicates that neutrophil-associated injury is more relevant in ischemic reperfusion injury than in permanent infarction.

Neutrophil Activation After MI Neutrophils are the first cell line to massively invade the heart after MI. They recognize so-called Bpathogen-associated molecular patterns,^ specific molecular patterns shared by groups of pathogens, such as lipopolysaccharides (LPSs) of bacteria or RNA of viruses [8] immediately. Those pattern recognition receptors are also activated by Bdanger-associated molecular patterns^ (DAMPs), molecules released from injured cells. DAMPs associated with MI include heat shock proteins [8], high-mobility group box 1 (HMGB-1), low-molecular hyaluronic acid, and fibronectin fragments [9]. DAMPs are sensed by pattern recognition receptors expressed on the surface or within the cytoplasm of neutrophils, other immune cells such as dendritic cells (DCs), and parenchymal cells including cardiomyocytes [10]. Especially, TLR2 and TLR4 play key roles in myocardial ischemia. Indeed, blockade of TLR4 [11] or TLR2 [12] could reduce infarct size in ischemic mice models (TLR signaling in post-ischemic inflammation is beyond the scope of this review; for a more detailed description, please refer to [13]). The complement system is also activated by DAMPs such as cardiolipin [14]. The classical lectin and alternative pathways are all implicated in pathogenesis of I/R injury [9]. Receptors for advanced glycation end products (RAGE) or TLR9 reacts with complexes of HMGB1 and DNA or nucleosomes [15]. Injection of HMGB-1 resulted in neutrophil accumulation, whereas HMGB-1-blocking antibodies inhibited neutrophil infiltration in LPS-induced lung injury [16]. TLR signaling converges on the activation of nuclear factor kappa B (NF-κB), a key signaling component fostering an inflammatory reaction. Both in I/R and permanent coronary ligation models, blockade of NF-κB attenuated myocardial injury and left ventricular remodeling [8]. Thus, neutrophils are activated after MI triggered by the innate immune system.

Myocardial Neutrophil Invasion After MI After the onset of myocardial ischemia, neutrophil-attracting CXC chemokines are rapidly and markedly upregulated. Those chemokines are immobilized to glycosaminoglycans on endothelial cell surfaces or in the extracellular matrix [17]. Two steps regulate neutrophil extravasation from vessels into the myocardium: First, neutrophils are captured on endothelial cells reversibly via interaction of selectin families on neutrophils and vascular cell adhesion molecules (VCAM) expressed on activated endothelial cells. In the second step, the firm arrest of neutrophil is mediated by chemokineinduced activation of CD18/CD11 integrin (LFA1, Mac-1, p150, p95) on neutrophils and expression of intercellular adhesion molecule (ICAM)-1/ICAM-2 on endothelial cells [18]. Several animal experiments and clinical trials tried to reduce neutrophil accumulation after MI by targeting neutrophils’ invasion signals. Monoclonal antibodies against Lselectin [19] and P-selectin were effective in lowering neutrophil accumulation and reducing myocardial necrosis within a few hours after reperfusion [20]. However, opposite effects have also been reported, e.g., no difference in myocardial injury in P-selectin devoid mice, in spite of marked reduction of leukocyte accumulation [21]. Antibodies to either all or one of the four isoforms of the CD11/CD18 integrin receptor reduce infarct size, improve coronary blood flow, improve left ventricular function, and decrease neutrophil infiltration in experimental ischemia reperfusion models [22]. This triggered clinical studies using an anti-CD11/18 antibody (Hu23F2G). However, Hu23F2G did not reduce infarct size in patients who underwent primary angioplasty [22]. The exact reason for the neutral outcome is unknown. Problems might include the effectiveness of the anti-CD18 antibody [23]. Thus, the reduction of neutrophil invasion does not seem to be a clinical effective therapy after MI. Potential Mediators of Neutrophil Mediated Damage Neutrophils release high amounts of reactive oxygen species (ROS) induced through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (respiratory burst) [24]. ROS can directly react with lipids, proteins, and DNA causing cell injury [25] and induce cytokine and chemokine release partially mediated by NF-κB. Inhibition of NADPH oxidase attenuated post-MI cardiomyocyte apoptosis and cardiac function in rabbit [26]. On the other hand, antioxidant treatment reduced microvascular density in the infarcted myocardium at day 7 post-MI, indicating that ROS promote angiogenesis and contribute to cardiac repair [27]. Granule components of neutrophils are also harmful. Myeloperoxidase (MPO), known as a diagnostic plasma marker of MI patients, is associated with increased long-

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term mortality in acute MI (AMI) patients [28]. Increased proteinase 3 [29] and neutrophil gelatinase-associated lipocalin (NGAL) [30] also predict adverse long-term prognosis and heart failure after MI. Neutrophils release matrix metalloproteinases (MMP) 8 and 9, which degrade the extracellular matrix. MMP-8 and MMP-9 are elevated in patients with left ventricular rupture after MI [31]. Inhibition of MMP-9 in mice attenuated LV dilation and collagen accumulation in infarcted area after coronary ligation [32] and promoted angiogenesis [33]. On the contrary, transgenic overexpression of MMP in macrophages improved left ventricular (LV) remodeling and reduced inflammatory responses in the LV infarct tissue and in isolated macrophages [34]. Macrophage-derived MMPs degrade the ECM to allow formation of new vessels [35]. Thus, the function of MMPs depends on the stage of healing after MI. MMP 8 and 9 released from neutrophils in the early stage after MI degrade extracellular matrix and intensify the inflammation. MMPs released from macrophage at a later stage support angiogenesis and beneficial remodeling. Collectively, neutrophils play a fundamental role in degrading necrotic tissue and accelerating pro-inflammatory cytokine production leading to clearance of debris by the following macrophages. However, a prolonged activity of neutrophils amplifies cardiomyocyte injury resulting in adverse cardiac remodeling. Neutrophil Extracellular Traps in MI Netosis is a form of neutrophil death different from apoptosis spreading Bneutrophil extracellular traps^ (Nets). Neutrophils release decondensed chromatin including toxic histones together with proteases [36]. Nets are an effective system to trap and kill bacteria and prevent expansion of local infection. However, Nets are also cytotoxic for host tissue and thrombogenic. Nets are involved in pathogenesis of thrombus formation of AMI patients [37] and in myocardial damage in mice after MI [38]. Activated platelets present HMGB-1 to neutrophil RAGE and commit them to Net generation [39]. Cleaving and clearance of Net chromatin by DNase1 administration revealed cardioprotective effects, resulting in subsequent improvement of cardiac contractile function in I/R mice model [38]. Indeed, inhibition of Net formation or rapid degradation might be a new attractive therapeutic option to reduce ischemic cardiac injury. Interaction of Neutrophils with Monocytes/Macrophages Monocyte differentiates into macrophages after migration from vessels inside tissues. Neutrophils and monocytes/ macrophage interact closely to facilitate recruitment,

phagocytosis, cytokine release, and resolution of inflammation in the heart (Fig. 1): 1. Patrolling monocyte and resident macrophages sense DAMPs and produce neutrophil-attracting cytokines and chemokines (TNF-α, IL-6, chemokine (CXC motif) ligand (CXCL)1, CXCL2) [40]. 2. Infiltrating neutrophils release granule contents (azurocidin, cathelicidin antimicrobial peptide (LL-37), and cathepsin G) promoting monocyte/macrophage recruitment [40]. Neutrophils also produce soluble IL-6 and IL-6 receptor complex which activates endothelial cells to express chemokine (C-C motif) ligand (CCL)2 and VCAM1 [41]. Macrophages and DCs secrete IL-23 which promotes IL-17 production by T cells. IL-17 stimulates granulopoiesis through increased G-CSF levels and neutrophil recruitment [42, 43]. 3. Neutrophil life span is short. Apoptotic neutrophils secrete Bfind me signal^ (lipid mediator such as lipoxins and resolvins, nucleotides) and Beat me signal^ (lysophosphatidylcholine) [17, 44] attracting scavenger cells. Lipid mediators, lactoferrin, and annexin A1 released from apoptotic neutrophil inhibit further influx of neutrophils but promote monocyte migration [45] into infarcted tissue. 4. Phagocytosis of apoptotic neutrophils can stimulate macrophages to produce the anti-inflammatory cytokines, transforming growth factor beta (TGF-β) and IL-10, and lipoxins and resolvins [40]. Thus, apoptosis of neutrophils plays a key role to operate negative feedback for continuous neutrophil migration, while phagocytic clearance of apoptotic neutrophil reprograms macrophages toward an anti-inflammatory phenotype which leads to resolution of inflammation. Prognostic Value of Peripheral Leukocyte Counts in Patients Total leukocyte count [46, 47], neutrophilia [48, 49], and neutrophil to lymphocyte ratio (NLR) [50] are associated with poor prognosis in patients with mixed etiology of HF. NLR is also correlated with cardiac function and exercise capacity in idiopathic cardiomyopathy (DCM) [51, 52]. The activated sympathetic nervous system [50], increased catecholamines, endogenous cortisol, and inflammatory cytokines seem to mediate those effects [53, 54]. In addition, splanchnic congestion and bacterial translocation activate monocytes to produce inflammatory cytokines such as TNF-α and IL-6 [54]. These cytokines also cause lymphocyte apoptosis and relative neutrophilia. Moreover, inflammatory cytokines trigger the activation of the sympathetic nervous system in the brain [55]. Patients with β-blockers have less tendency of this leukocyte redistribution [53].

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Fig. 1 Inflammatory cells and infarct healing. Patrolling monocyte and resident macrophage recruit neutrophils via DAMPs. Neutrophils promote pro-inflammatory monocyte recruitment. Neutrophils and Ly6C high monocytes/M1 macrophages cooperate to amplify the inflammation releasing cytokines (TNF-α, IL-6, IL-1b), degrade, and phagocyte necrotic debris, but damage also healthy tissue. Apoptotic

neutrophils inhibit further neutrophil influx. Macrophage phagocytosis of apoptotic neutrophil and Treg cells accelerate the shift of monocyte/ macrophage phenotype from pro-inflammatory to anti-inflammatory, leading to angiogenesis and collagen deposition. DAMPs damageassociated molecular patterns, Treg cell regulatory T cell, VEGF vascular endothelial growth factor

Monocytes and Macrophages After MI

and Ly-6Clow monocyte correspond to CD14+CD16− and CD14+CD16+ monocyte in humans with regard to their cytokine profile and function in healing [60]. Indeed, a recent autopsy study confirmed monocyte kinetics after MI not only in the animal but also in the human heart; that is, CD14 + CD16 − monocytes accumulate early, whereas CD14+CD16+ monocytes dominate later after MI [61]. Ly-6Chigh monocytes are recruited to the myocardium through interaction with MCP-1/CCR chemokine/chemokine receptors [58, 62], followed by increased endothelial expression of adhesion molecules such as selection and VCAM1 [63]. Ly-6Chigh monocytes express inflammatory cytokines (TNF-α, IL-1β, MPO, MMPs) and proteolytic mediators (cathepsins, plasminogen activator urokinase) promoting digestion of infarcted tissue and removal of necrotic debris. Ly-6Clow monocytes express anti-inflammatory cytokines (IL-10, TGF-β) and VEGF, mediating myofibroblast accumulation, angiogenesis, and deposition of collagen, necessary for granulation tissue during the resolution of inflammation [58] (Fig. 1)

Macrophages play a central role in wound healing or tissue regeneration [56] and, therefore, are essential to heal tissue damage after ischemic injury in general. Function and balance of inflammatory M1 and anti-inflammatory M2 macrophages determine the direction of remodeling (Fig. 1) Pro-Inflammatory and Anti-Inflammatory Phenotypes of Monocytes/Macrophages After MI Monocytes/macrophages are the first cells recruited to the myocardium after ischemia onset. A recent study using intravital microscopy demonstrated that monocyte recruitment even outpaces neutrophils within the first 30 min [57]. This is followed by a massive monocyte/macrophage infiltration after the influx and resolution of neutrophils. Subsets of monocyte are stratified in two types in mice, pro-inflammatory phenotype (Ly-6Chigh monocyte), which dominates the first phase with a peak around day 3 (phase I), and anti-inflammatory phenotype (Ly-6Clow monocyte), which comes later during days 4–7 (phase II) post-MI [58]. Macrophages can also be subdivided into a pro-inflammatory M1 and a healing M2 phenotype. This biphasic monocyte/ macrophage response has been shown in both permanent coronary ligation [58] and I/R [59] models. Ly-6Chigh

How Does the Phenotype of Monocytes/Macrophages Affect Myocardial Remodeling? Apolipoprotein E−/− mice have a Ly-6Chi blood monocytosis. Experimental MI in apolipoprotein E−/− mice led to adverse

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outcome together with an increased myocardial infiltration of inflammatory Ly6Chi monocytes and delayed transition to the Ly-6Clow monocyte phase [64]. On the contrary, silencing CCR2 during the first week after MI reduced recruitment of Ly-6Clow monocytes and improved ejection fraction after MI [65]. In clinical studies, monocytosis after MI was associated with LV dysfunction and aneurysm formation [66]. Additionally, the peak peripheral blood levels of CD14+CD16− monocytes, but not CD14+CD16+, were negatively associated with the extent of myocardial salvage and recovery of LVEF [66]. Monocyte and macrophage depletion led to increased mortality and also high incidence of left ventricle thrombus formation, possibly caused by impaired endocardial healing. Moreover, this insight was confirmed clinically. Patients with a LV thrombus had a relative decrease of CD14+CD16+ monocyte subsets in the peripheral blood after MI [67]. Monocytes/macrophages can be experimentally depleted by intravenous injection of clodronate-loaded liposomes. Monocyte/macrophage depletion during inflammatory phase I resulted in impaired removal of necrotic debris, whereas depletion during the anti- inflammatory phase II leads to decreased deposition of collagen and a reduced number of microvessels [58]. Notably, silencing interferon regulatory factor 5 (IRF5) using siRNA, which regulates polarization toward inflammatory M1 macrophages in MI, shifted the macrophage phenotype from M1 to anti-inflammatory M2 in the heart and attenuated post-MI HF [68•]. Regulatory T cells (Treg cells) have recently been recognized to be involved in the monocyte/macrophage phenotype switch after MI. Genetic or antibody-mediated (anti-CD25) ablation of Treg led to M1 macrophage polarization together with prolonged inflammation and adverse cardiac remodeling after MI. Therapeutic Treg cell activation induced an M2 macrophage differentiation associated with beneficial outcome, myofibroblast activation, and increased expression of monocyte/macrophage-derived proteins fostering wound healing [69]. Hence, these observations indicate that appropriate healing requires a well-coordinated biphasic monocyte response where, experimentally, the pro-inflammatory phase last only for a couple of weeks in the mouse. A prolonged proinflammatory phase seems to be maladaptive [70, 71].

Source of Macrophage Recruitment After MI Although the major source of blood cells under steady-state conditions is the bone marrow, monocytes/macrophages immediately influx the heart from the spleen after MI [72]. One day after coronary ligation in mice, about half of the monocytes emigrate from the splenic reservoir, whereas the monocyte number in the bone marrow is unchanged [60]. This monocyte expulsion is dependent on angiotensin II [60], but

not on CCR2 chemokine receptor signaling that is crucial for monocyte mobilization from the bone marrow. ACEI reduces the release of splenic monocytes and attenuated LV remodeling 3 weeks after coronary ligation in mice [73]. In accordance, numbers of CD14+ cells in the spleen of AMI patients were profoundly decreased by autopsy specimens [61]. The life cycle of monocytes at the site of inflammation is as short as 20 h in mice. Therefore, the number of monocytes/macrophage infiltrating the infarcted heart is mainly regulated by recruitment and death balance [74]. To allow an adequate number of infiltrating monocytes/macrophages, splenic hematopoiesis is stimulated after MI [74]. Activation of sympathetic nervous system via beta-3 adrenoreceptors on niche cells in the bone marrow increases hematopoietic progenitor cell mobilization to the spleen [75], where monocytopoiesis occurs under regulation by IL-1β [73]. Splenectomy on day 1 or day 3 after coronary ligation reduced the numbers of monocytes/macrophages recruited to the infarct and led to impaired wound healing after MI [74]. In contrast, splenectomy reversed pathological cardiac remodeling and inflammation when performed 8 weeks after coronary ligation [76•]. Splenocytes adoptively transferred from HF to healthy mice homed to the heart and induced long-term LV dilation and dysfunction in naive recipients. Splenocyte sorting at transfer time revealed reduced pro-inflammatory monocytes (CD11+/F4/80+) and markedly increased DCs (CD11c+). These findings indicate the significance of spleen as a supplier of inflammatory monocytes and regulator of cardiac inflammatory remodeling after MI. ACEI and β-blockers are also immune modulators in HF treatment. Especially early after MI, these drugs might be relevant in attenuating inflammatory monocyte hematopoiesis, splenic release, and influx to the myocardium.

Conclusion Neutrophils play a pivotal role after MI. Spread of DAMPs sensed by innate immune cells triggers the cascade. Neutrophils are activated and recruited by cytokines and chemokines, release abundant cytokines and granule proteins, and generate ROS, which modify molecules or degrade necrotic tissue directly. Nets are involved in pathophysiology in this early inflammatory phase after MI. The initial inflammatory response leads to the recruitment of pro-inflammatory, followed by pro-healing monocytes/macrophages. Macrophages/monocytes are mainly recruited from the spleen. However, the translation of the results in a clinical context is difficult. This is due to the fact that inflammation per se is an adequate response to tissue injury. Only exaggerated or inadequate inflammation is pathophysiologically relevant. Thus, not only a general reduction of the inflammatory burden

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seems to be useful, but also a specific, timely restricted approach. There are promising new tools to do this: for example, siRNA application might be a valuable tool for this purpose. As mentioned above, silencing CCR2, a chemokine receptor crucial for attracting Ly6C high monocyte during the first week after MI, improved subsequent ejection fraction [65]. Likewise, macrophage polarization from M1 to M2 by silencing IRF5 elegantly attenuated HF after MI [68•]. Progress of imaging modalities such as intravital microscopy or iron oxide nanoparticle MRI [77, 78] made it possible to visualize immune cell kinetics and function and may thus help us to guide our therapeutic approaches. Next steps for successful immune modulation will come from selection of appropriate therapy for appropriate patients at an appropriate time point with excessive adverse inflammation. Compliance with Ethics Guidelines Conflict of Interest Hisahito Shinagawa declares that he has no conflict of interest. Stefan Frantz has received financial support through a grant from the Federal Ministry of Education and Research (BMBF). Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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