Mechanisms of cardioprotection via modulation of the immune response

Abstract

Both morbidity and mortality as a result of cardiovascular disease remain significant worldwide and account for approximately 31% of annual deaths in the US. Current research is focused on novel therapeutic strategies to protect the heart during and after ischemic events and from subsequent adverse myocardial remodeling. After cardiac insult, the immune system is activated and plays an essential role in the beginning, development, and resolution of the healing cascade. Uncontrolled inflammatory responses can cause chronic disease and exacerbate progression to heart failure and therefore, constitute a major area of focus of cardiac therapies. In the present overview, we share novel insights and promising therapeutic cardioprotective strategies that target the immune response.

Introduction

Cardiovascular disease (CVD) still accounts for 31% of all annual deaths in the US, of which ~20% are patients <65 years of age [1]. These statistics comprise patients with hypertension and chronic heart failure (HF), including myocardial infarction (MI), angina pectoris, and stroke. Cardiac injury induces cell death and/or tissue damage that stimulates an inflammatory response to remove cell/tissue debris [2]. This inflammatory response includes activation of toll-like receptor (TLR)-mediated pathways, complement system cascade, and generation of reactive oxygen species [3]. These, in turn, induce nuclear factor kappa B (NFκB) activation and upregulate the synthesis of chemokines and cytokines, which respectively stimulate recruitment of inflammatory leukocytes into the myocardium and promote adhesive interactions between leukocytes and endothelial cells, resulting in the infiltration of inflammatory cells into the site of injury [2–4].

Numerous reports show that the immune response elicited by cardiac ischemia and injury has an important role in CVD and progression to HF. For the last 20 years, research and ensuing clinical trials addressing modulation of the inflammatory response have focused mainly on immune-cells, such as leukocytes [2,3], decrease/inhibition of oxidative stress [5], and the use of anti-inflammatories [6]. Nevertheless, patient mortality and morbidity remain significant. For example, the use of immunosuppressive drugs, such as corticosteroids, has no effect on MI risk ratio or reduction of patient mortality [7]. Therefore, treatment with broad spectrum immunosuppressive drugs is not an adequate therapy for all CVD patients and innovative (personalized) immune-targeted therapeutics have not yet emerged in the clinical setting. In this short review, we will outline cardioprotective mechanisms that target the immune system (innate and adaptive) and are currently promising novel therapeutic strategies.

Innate immune response

Innate immunity is a pre-programmed (non-specific) first-line of defense, classically mediated by myeloid-derived cells that upon injury produce cytokines and activate the complement system. The innate immune-system plays a crucial role in the initiation and progression of the subsequent healing cascade.

Toll-like receptors

TLRs are transmembrane proteins, members of the family of pattern recognition receptors, involved in innate and adaptive immune responses in the identification of pathogens and in sensing endogenous danger-associated molecular patterns released from necrotic or dying cells [8]. The activation of TLRs triggers a downstream signaling cascade comprising activation of transcription factors, such as activator protein 1 and NFκB, followed by secretion of pro-inflammatory chemokines and cytokines, recruitment of phagocytes, and activation of the complement system (Figure 1) [9]. TLRs are expressed in innate immune cells and have also been identified in several cardiovascular cells, including cardiomyocytes and endothelial cells [10]. This suggests that TLR signaling could be important in the development of myocardial diseases and therefore, TLRs present a valuable therapeutic target. Of the TLR family, TLR4 is the most studied, and it is expressed in cardiac cells ~10-fold higher than most other TLRs [8]. TLR4 expression is significantly higher in patients with atrial fibrillation and is an independent predictor of atrial fibrillation recurrence [11]. Metformin, a first-line medication for type 2 diabetes, displays potential cardioprotective roles during lipopolysaccharide-induced inflammatory responses by attenuating TLR4 activity [12^••]. Ischemia-reperfusion (I/R) injury in hearts could also be treated with the application of TLR4 antagonists, since rats treated with such drugs demonstrated a decrease in infarct size [13]. In addition, the administration of the tricyclic anti-depressant amitriptyline curiously improved LV pressure recovery in I/R-induced rats [14], and moderate levels of tricyclics proved to alter TLR4 levels [15]. TLR4 signaling may also play an important role in angiotensin (Ang) II-induced hypertension. Crosstalk between AngII and TLR4 within the brain increased NFκB activity and sympathetic impulses contributing to hypertension progression and cardiac dysfunction in rat models [16]. In summary, TLRs are novel candidate molecules that connect myocardial injury and circulating inflammatory mediators, and therefore uncover potential therapeutic approaches to model the immune system.

Figure 1.

Myeloid differentiation primary-response protein 88 (MyD88)-dependent mechanism of Toll-like receptor (TLR)-4 activation of pro-inflammatory responses. TLR-4 antagonists can block the inflammatory cascade and provide cardioprotective effects. AP-1: activator protein-1; DAMPs: danger-associated molecular patterns: IkB, inhibitor of kB; JNK: c-Jun-N-terminal kinase; NFkB: nuclear factor kappa B.

Macrophages

During pathological cardiac remodeling and healing, both resident and circulating macrophages play crucial cardioprotective roles. In the inflammatory phase that follows cardiac injury, Ly-6Chi monocytes are recruited from the bone marrow and spleen to help resident macrophages remove dead/damaged tissue and cells, as well as, produce the necessary enzymes to facilitate extracellular matrix remodeling and vascularization [17]. Several subsets of cardiac macrophages have been defined; however, for the purpose of this review, we will only refer to the M1 and M2 phenotypes. Classically activated macrophages (M1, Ly-6C^hiCD206⁻CD204⁻) associate with pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS)-derived nitric oxide, tumor necrosis factor (TNF)-α, and interleukin (IL)-12 [18]. Secreted IL-12 further activates CD4 T-cells promoting the pro-inflammatory phenotype [17]. M1 macrophages display potent phagocytotic properties necessary to clear necrotic tissue [19]. Upon ingestion of apoptotic cells, macrophages release anti-inflammatory cytokines promoting a shift in the macrophage population towards a reparative phenotype that may restrain inflammatory injury and attenuate adverse cardiac remodeling [20]. Alternatively activated macrophages (reparative/M2, Ly6C^loCD206⁺) express IL-10, chitinase 3-like 3, and resistin like-beta and upregulate arginase-1 and CD206 [18]. M2 macrophages inhibit CD4 T-cell and granulocyte activity and display anti-inflammatory properties [17]. Interestingly, targeted depletion of either M1 or M2 population with clodronate liposomes leads to impaired cardiac healing evidenced by increased left ventricular dilation, reduced vascularization, and increased mortality post myocardial injury [21]. Several mechanisms have been reported that exert cardioprotective effects by promoting M2 macrophage polarization. In an I/R MI mouse model, a selective A2B adenosine receptor (A2BR) agonist reduced tissue injury by increasing phosphorylated Akt (p-Akt) levels [22]. The majority of p-Akt was colocalized to CD206⁺ cells, suggesting that A2BR activation promotes M2 polarization, and therefore, an anti-inflammatory response. Db/db diabetic mice that received fibroblast growth factor-9 treatment for two weeks post-MI exhibit reduced monocyte infiltration, increased M2 macrophage differentiation and anti-inflammatory cytokines (IL-10 and IL-1RA), and improved cardiac function [23^••]. Similarly, Miao et al. showed that hydrogen sulfide supplementation, in a mouse MI model, ameliorated pathological remodeling and cardiac dysfunction by enhancing M2-polarized macrophage levels at the early stage of MI [24]. When using cardiosphere-derived cells (CDCs) de Couto et al. demonstrated that infiltrating macrophages can be primed to acquire a cardioprotective phenotype in ischemic heart and mitigate ischemic injury through activation of an anti-apoptotic program in cardiomyocytes [25^••]. Unexpectedly, while the expression of M1 markers was decreased they did not observe an increase in M2 markers. These results highlight the heterogeneity of macrophage populations and raise important questions related to treatments’ mode of action on macrophage phenotype, time of intervention, and long-term effects.

Adaptive immune response

Adaptive immune responses can be divided into two broad classes: antibody responses and cell-mediated immune responses, which are carried out by B-cells and T-cells, respectively. While there is some literature on the experimental use of bone-marrow derived B-cells for treatment of cardiac dysfunction after ischemia [26], the majority of current research is focused on T-cells. Recent studies suggest that regulatory T-cells (T_regs) can decrease the infiltration of pro-inflammatory cells in injured hearts and prevent chronic inflammation that could lead to cardiac failure. According to Li et al., diabetic patients present overstimulation of protein kinase C (PKC)-θ, a member of the protein kinase super-family, which recruits and activates T-cells to secrete IL-1 and IL-6 [27]. These pro-inflammatory cytokines can cause deterioration of the zona occludens protein 1, increasing tight junction permeability in cardiac endothelial cells, which would lead to increased cell extravasation. Using a specific PKC-θ inhibitor in a mouse type 1 diabetic cardiomyopathy model, Li et al. preserved tight junction integrity and reduced intramyocardial T-cell infiltration, which led to decreased fibrosis and improved cardiac function [27]. Similarly, fingolimod (FTY720), an immunosuppressant analogue of sphingosine used to treat multiple sclerosis, may act as an anti-fibrotic agent for diabetic patients. After chronic administration of FTY720 in a diabetes-induced mouse model, it was noticed a decreased number of T-cells in the myocardium with an associated reduced expression of transforming growth factor (TGF)-β and fibrosis [28^•]. Scavenger receptor class B member 1 (SR-BI) can prevent atherosclerosis development. Mouse models with SR-BI-deficient and hypomorphic ApoE (ApoeR61^h/h/SRBI^−/−) under long-term treatment with FTY720 presented an increase in their survival rate in relation to non-treated animals [29]. The study suggested that FTY720 may be involved in T_reg activation since several tissues from the treated animals presented an increase in T_reg cells; however, without a reduction in coronary atherosclerosis level, compared to non-treated animals. Imbalance between plasma levels of pro-inflammatory T helper 17 cells (Th17) and anti-inflammatory IL-10 has been reported in patients with heart failure [30]. Zhang et al. demonstrated that chronic HF-induced rats treated with catechin, an active ingredient of green tea, presented a reverse in the balance of Th17/IL-10 plasma levels [31]. Animals treated with catechin also presented higher levels of TGF-β, a factor that causes T_reg differentiation via FoxP3, compared to non-treated rats. A group from the Fujian Medical University proposed that statins may play a pleiotropic role in cardioprotection by modulating T_reg cells on top of its HMG-CoA reductase inhibition effect [32]. This laboratory also showed that low doses of N,N-dimethylsphingosine (DMS), a natural sphingosine derivative, could be involved in the recruitment of T_regs via the PI3K/Akt pathway after an ischemic event [33^•]. PI3K/Akt is activated during ischemia, and mice pre-treated with low portions of DMS showed diminished mRNA values of TNF-α and IL-1β and decreased neutrophil infiltration in the myocardium after I/R injury.

Of note, aging significantly and inversely correlates with the cardioprotective role of T-cells. With age the immune system changes, a process commonly referred to as immunosenescence. The thymus size and functionality naturally decrease with age and this directly affect T-cell maturation [34]. In the elderly, a decrease in the pool of naïve T-cells often results in an increased susceptibility to pathogenic attack and the subsequent development of cardiac dysfunction [35]. Large cohort studies have proven that Influenza vaccination reduces the number of cardiomyopathy hospitalizations by activating pre-existing memory T-cells [36]. This emphasizes the important role of T-cells in cardioprotection and offers an attractive target for treatment of cardiac dysfunction through modulation of the adaptive immune response.

Ischemic pre-conditioning and post-conditioning

Ischemic pre-conditioning (IpreC) is considered a potent endogenous mechanism to protect the myocardium from I/R injury. IpreC is defined as an increased tolerance to I/R induced by a previous sublethal period of ischemia, and can result in acute cardioprotection (classical preconditioning, lasts 1–3 h) followed by a delayed phase of protection (late preconditioning, starts 24 h after initial ischemia and lasts up to 72 hours) [37,38]. IpreC is thought to evoke cell survival programs in the heart via the activation of innate/intrinsic cytoprotective programs, such as G-protein coupled receptor signal transduction pathways and by reducing the burst of reactive oxygen species generated from inflammatory cells [39]. Furthermore, IpreC can result in T_reg aggregation followed by reduced infiltration of inflammatory cells in the ischemic area [40]. IpreC in vitro studies using cardiomyocytes, exposed to hypoxic conditions for 5 hours and subsequently treated with gases (xenon and isoflurane) and calcium sensitizers (levosimendan), also displayed improved survival rates as compared to non-treated cells [41]. The xenon preconditioned group showed an elevated level of vascular endothelium growth factor, indicative of increased vasculogenesis and angiogenesis activities. Wong et al. subjected rats to I/R injury and found that IpreC with morphine derivatives, targeted at opioid receptors, resulted in reduced infarct size in relation to the area at risk for injury [42]. Even though IpreC has emerged as a powerful strategy to delay myocardial cell death during ischemia, its clinical applicability has shown slow progress and is still being evaluated [43]. Nonetheless, the IpreC activated signaling pathways are current targets of interest.

The potential for therapeutics involving cardioprotective properties of ischemic post-conditioning (IpostC) is currently the topic of a myriad of investigative activities. Treatments involving IpostC have included a variety of pharmaceuticals, such as opioids and chloroquines. Their cardioprotective effects are thought to result from reduced release of inflammatory mediators, which minimizes I/R pathological changes in myocardial cells. Recently, studies conducted by Zheng et al. demonstrated that berbamine IpostC is cardioprotective in rats, and cardioprotection was dose dependent [44]. This determination was based on measurement of infarct size, left ventricular function, and cell survival ex vivo; as well as, cardiomyocyte survival, contraction and mitochondrial membrane potential in isolated cells. Also recently, and perhaps most clinically significant, is a study conducted by Kanazawa et al. which utilizes allo CDCs post reperfusion for the IpostC treatment of pigs after MI [45]. This study revealed that CDC-induced IpostC resulted in reduced infarct size and microvascular obstruction, due to attenuation of myocardial remodeling when compared to control animals. Considering the variety of approaches and diverse experimental designs, which all appear to have some beneficial effects in decreasing infarct size and/or increasing wound healing activities, it is evident that further studies in this area are both necessary and clinically pertinent due to the overall implications in improved patient outcomes.

Estrogen

Similar to other physiological systems, the immune system also demonstrates significant sex differences. The reasons for these differences in immune responses are not completely understood, but potentially include differences in sex hormones, such as estrogen. In the last decades, it has been observed that the risk of CVD appears to be inversely related to endogenous estrogen levels, as a decrease in estrogen secretion may cause hypertension and metabolic dysfunctions, such as insulin resistance, which are associated with CVD development [46]. Estrogen has been shown to regulate the numbers and functions of innate and adaptive immune cells (comprehensively reviewed in Ref. [47]). Furthermore, estrogen therapy in association with vildagliptin (a dipeptidyl peptidase-4 inhibitor) ameliorates cardiac mitochondrial dysfunction by reducing oxidative stress, in estrogen-deprived insulin-resistant female rats [48]. One possible mechanism whereby estrogen may act in cardioprotection is by downregulating TNF-α production in cardiac inflammatory cells. TNF-α neutralization by estrogen prevents inflammatory cells from affecting fibroblast functionality improving cardiac remodeling with appropriate collagen reposition in volume overload-induced rats [49]. The G-protein estrogen receptor (GPER) is present in both females and males, and it can be considered as a therapeutic target for CVDs. GPER activation by agonist G-1 not only decreased cardiac inflammation, but also improved heart’s mechanical performance in an I/R injury male rat model [50]. In view of these findings, gender differences should be considered a critical factor for future research design and in therapeutic approaches involving CVD patients [51].

Discussion

Scientific discoveries reveal daily the complexities and similarities between physiological systems, and how such correlations confound the development of more precise medications. Yet, existing drugs aimed at other disorders are recently demonstrating unexpected beneficial effects in CVD treatments through modulation of the immune system. For example, metformin, initially used for diabetes treatment, may also preserve cardiac function by blocking TLR4 activity [12^••]; hydrogen sulfide could ameliorate cardiac remodeling if administrated at early stages of MI by stimulating M2 macrophage polarization [24]; and IpreC with morphine proved to reduce infarct size by blocking opioid receptors [42]. In addition to having a cardioprotective effect, the innate and adaptive immune activation can also participate in the development of cardiopathies. This ambiguous outcome can occur by unregulated inflammatory responses caused by diabetic cardiomyopathy, ischemic events, atherosclerosis, and other types of CVD. This review emphasizes promising mechanisms by which the immune system may provide cardioprotection upon myocardial injury. Nonetheless, time of intervention (and dosage) may have different effects in such a complex and intricate system, leading to diverse clinical outcomes. Thus, novel therapeutic approaches that target modulation of the immune system to result in a more balanced inflammatory response upon myocardial ischemia are still warranted.