Interleukin 4: Its Role in Hypertension, Atherosclerosis, Valvular, and Nonvalvular Cardiovascular Diseases

Abstract

Hypertension is one of the major physiological risk factors for cardiovascular diseases, and it affects more than 1 billion adults worldwide, killing 9 million people every year according to World Health Organization. Also, hypertension is associated with increased risk of kidney disease and stroke. Studying the risk factors that contribute to the pathogenesis of hypertension is key to preventing and controlling hypertension. Numerous laboratories around to globe are very active pursuing research studies to delineate the factors, such as the role of immune system, which could contribute to hypertension. There are studies that were conducted on immune-deficient mice for which experimentally induced hypertension has been ameliorated. Thus, there are possibilities that immune reactivity could be associated with the development of certain type of hypertension. Furthermore, interleukin 4 has been associated with the development of pulmonary hypertension, which could lead to right ventricular remodeling. Also, the immune system is involved in valvular and nonvalvular cardiac remodeling. It has been demonstrated that there is a causative relationship between different interleukins and cardiac fibrosis.

Background

Humans with elevated interleukin (IL) 4, such as is common with aging,¹ asthma,² and scleroderma,^3,4 are at high risk of cardiac fibrotic remodeling and dysfunction.^5,6 Certain alleles, genotypes, and haplotypes in IL-4 genes are overexpressed in some patients with ischemic heart failure (HF).⁷ Likewise, Balb/c mice have high IL-4 and cardiac fibrosis.⁸ We and others have demonstrated a causative relationship between IL-4 and cardiac fibrosis.^9,10 In the hearts of Balb/c mice, there were a substantial number of CD4⁺ T cells, one of the sources of IL-4, compared to IL-4^−/−, while wild-type (WT) mice displayed an accumulation of mast cells, macrophages, and bone marrow (BM)-derived fibroblasts in the heart and an elevation in cardiac monocyte chemoattractant protein 1 (MCP-1) levels.¹⁰ Interleukin 4 also induces MCP-1 production in cardiac endothelial cells and fibroblasts.¹¹ Reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidases (Nox2 and Nox4) contribute to cardiac fibrosis.^12,13 Both Nox2 and Nox4 are constitutively expressed, and IL-4 strongly induces the ROS-mediated expression of the transcription factor activator protein (AP) 1 and collagen-1a in cardiac fibroblasts.^14,15 The number of circulating BM-derived fibroblast progenitor cells (BMFPCs) was significantly increased in patients with hypertension having heart disease.¹⁶ Elevated levels of IL-4 are seen in patients with hypertension⁵ and experimental animal models.^9,17

Cellular Sources of IL-4 in the Heart

Interleukin 4 is produced by T-helper type 2 (Th2) cells¹⁸ and several cell types of the innate immune system including eosinophils,¹⁹ mast cells, and basophils.^20,21 However, there are still several unknowns with regard to contribution of each cell type in production of cytokines during acute and chronic inflammatory settings and in different tissue/organ. While Th2 cells and eosinophils are the cellular sources of IL-4 in asthma, IL-4 is produced by Th2 cells, basophils, and eosinophils in response to helminth infections.²²

Interleukin 4 Signaling Pathway

Interleukin 4 binding lead to the formation of 2 types of receptor complexes, namely, IL-4Rα and IL-13Rα1. Activation of IL-4Rα triggers the recruitment of the IL-4Rγ chain to form the type I receptor complex, whereas that of the IL-13Rα1 activates the type II receptor complex (Figure 1). Interleukin 13 engages in the c recruitment of the IL-4Rα chain.²³ Binding of IL-4 to the receptors and heterodimerization leads to the activation of the Janus kinase family of enzymes (JAKs). Activated JAKs phosphorylate signal transducer and activator of transcription (STAT) 6, which then migrates to the nucleus and binds to the promoters of the IL-4 and IL-13, such as those associated with Th2 cell differentiation, airway inflammation, airway hyperresponsiveness, and mucus production in the setting of asthma.²⁴

Figure 1.

Diagram depicting the cellular signaling linked to the interleukin (IL)-4/IL-13 receptor(s) activation leading to cardiac fibrosis and dysfunction. Both IL-4 and IL-13 signal via the IL-4Rα a component of the type I (IL-4Rα and γc) and type II receptors (IL-4Rα and IL-13Rα1). Profibrotic effects of IL-4/IL-13 might be mediated not only through signal transducer and activator of transcription 6 (STAT-6) but also reactive oxygen species (ROS) signals via both type I and II receptor pathways, whereas IL-13 signals only via the type II IL-4R. Phosphorylated STAT-6 dimerizes, migrates to the nucleus, and binds to the promoters of the IL-4 and IL-13 responsive genes, such as those associated with ROS-dependent AP-1 and collagen-1 (Col)a gene, leading to cardiac fibrosis and dysfunction (Adapted from Oh et al²⁴).

Role of IL-4 in Hypertension

Hypertension affects about 20% of adults worldwide, and there are many factors that could contribute to its development.²⁵ The role of some immune cytokines has been proposed for the possible mechanisms leading to hypertension and associated target organ damages and dysfunction. Particularly, one study showed that the administration of anti-IL-4 antibody suppresses hypertension in NZBW rats.²⁶ There are other cytokines that have been investigated for their role in the pathogenesis of hypertension. Studies have shown that deficiency of IL-1r in mice,²⁷ IL-6,²⁸ and IL-10^14,29 ameliorate the hypertension resulting from angiotensin II (Ang II) infusion. These studies illustrate that the immune reactivity in many autoimmune diseases may be the driving force for hypertension.

In addition, our recently published results demonstrate that high levels of IL-4 make the heart more susceptible to increased afterload in Ang II-induced hypertension, resulting in dilated cardiomyopathy.¹⁰ It remains unclear whether IL-4^−/− or IL-4Ra^−/− mice are protected against chronic hypertension and associated organ damage. A retrospective study has shown that women who had preexisting asthma diagnoses have also preexisting diagnoses of chronic hypertension, resulting in increased risk of gestational hypertension and preeclampsia.³⁰ A different study showed that the use of short-acting b2-agonists during pregnancy reduced gestational hypertension.³¹ This could illustrate that maintaining good control of asthma could prevent the development of severe hypertension. One study showed that there is a positive correlation between IL-4 serum level and uncontrolled asthma, but it was not significantly different from patients with controlled asthma.³² However, another explanation is that b2-receptors lower blood pressure via the vasodilatory effects of b2-receptor agonist on the vascular smooth muscle of the peripheral vasculature.³¹ Another retrospective cohort study found that persistent asthma increased the risk of chronic kidney disease, which was independent of other established risk factors such as obesity, diabetes, and hypertension.³³ The mechanism for how asthma increases kidney disease is not clear, but increased IL-4 could be a possible mechanism given that previous research has shown to induce hypertension. Other immune-mediated disease, such as inflammatory bowel disease, has been shown to increase the risk of coronary artery disease and asthma.³⁴ One study showed that there was a correlation between elevated IL-1 in first trimester and preterm preeclampsia,³⁵ but it did not show any correlation between other immune markers such as IL-4. However, a recent study showed that the infusion of IL-4 in reduced uterine perfusion pressure in rats improved mean arterial pressure and decreased inflammatory markers.³⁶

On the other hand, a multicenter AIDS cohort study indicated that there is a lower prevalence of systolic hypertension in untreated HIV-positive patients with low numbers of CD4+ cells than in treated HIV-positive patients or uninfected controls.³⁷ This shows hypertension could be ameliorated in an immune immunosuppression state. Nevertheless, further studies are needed to better delineate the role of IL-4 in the various types of hypertension. Table 1 illustrates how different experimental animal models of hypertension responded to selected immunodeficiency.

Table 1.

Immunodeficiency Effects on Hypertension.

Hypertension Model	Experimental Models	MAP	Reference
Ang II infusion	IL-1R knockout mice	WT = 180 mm Hg; IL-1R−/− = 165 mm Hg	27
NZBFI rats	Anti-IL-4 antibodies	Hypertension ameliorated	26
Ang II infusion	IL-6 knockout mice	WT = 160 mm Hg; IL-6−/− = 134 mm Hg	28
Ang II infusion	IL-10 knockout mice	Unchanged blood pressure	29
Ang II infusion	IFN-γ knockout	WT = 170 mm Hg; IFN-γ−/− = 148 mm Hg	38
Ang II infusion	TNF-α knockout	WT = 151 mm Hg; TNF-α−/− = 113 mm Hg	39
Ang II infusion	IL-10 knockout mice	Hypertension unchanged	40
Ang II infusion	IL-17 knockout mice	Hypertension ameliorated	29

Abbreviations: SBP, systolic blood pressure; MAP, mean arterial pressure; WT, wild type; Ang II, angiotensin II; IL-1R, Interleukin-1 receptor; IFN-γ, interferon γ; TNF-α, tumor necrosis factor α.

Role of IL-4 in Cardiovascular Diseases

Interleukin 4, a typical Th2 cytokine,⁴¹ promotes tissue fibrosis in diseases involving the lung,⁴² skin,³ and liver.⁴³ Inhibition of tissue fibrotic remodeling in the lung and skin due to IL-4 has been demonstrated in various animal studies.^34,44 Widely used in Japan by patients with asthma,^45,46 suplatast tosilate inhibits airway fibrosis by lowering levels of IL-4 in clinical applications.^2,47,48 Elevated IL-4 is closely associated with cardiac fibrotic remodeling and dysfunction in both experimental animals^17,49,50 and humans^5,6 with hypertension, advancing age, and postviral myocarditis. Strong IL-4 gene polymorphism has been reported in patients with ischemic HF.⁷ A profibrotic role of IL-4 in the heart has recently been demonstrated in a loss-of-function study using a hypertension mouse model⁹ as well as our study employing WT Balb/c (with high systemic and cardiac IL-4) and IL-4^−/− mice.¹⁰ Thus, Balb/c is a perfect translational mouse model in which it is unnecessary to artificially stimulate IL-4 or infuse exogenous IL-4 to increase circulating and local IL-4. In addition, we reported for the first time that IL-4 induces procollagen gene expression and robust collagen production through the activation of the STAT6 pathway in cardiac fibroblasts.¹⁰ As a result, the causative relationship between IL-4 and cardiac fibrosis has been well established. However, the underlying mechanism of IL-4-induced cardiac fibrosis is still not fully understood. Questions that remain to be answered regarding the role of IL-4 in the cardiovascular system include but not limited to (1) the cellular and molecular responses of IL-4-reactive cells in the heart during the development of IL-4-induced fibrotic cardiomyopathy and (2) the cellular sources of local IL-4 and elucidation of the role of IL-4 in the recruitment of BMFPCs to the heart and its impact on cardiac fibrosis. Figure 1 illustrates the possible cellular mechanisms through which IL-4 lead to cardiac disease and cardiac fibrosis.

Role of IL-4 in the Recruitment of BMFPCs to the Heart and its Consequent Impact on Cardiac Fibrosis

The importance of BM-derived fibroblasts in the development of cardiac interstitial and perivascular fibrosis has recently been reported in mouse models of ischemic cardiomyopathy,⁵¹ the chronically failing heart,⁵² and Ang II hypertension.⁵³ Importantly, a recent study showed that the number of circulating BMFPCs was significantly increased in patients with hypertension having heart disease.¹⁶ Elevated levels of IL-4 are seen in patients with hypertension⁵ and experimental animal models.^9,17 These results indicate that elevated IL-4 may promote recruitment of BMFPCs to the heart, significantly contributing to IL-4-induced fibrotic cardiomyopathy, but more studies are needed to confirm this hypothesis. The BMFPCs use different chemokine ligand–receptor pairs for tissue homing. Indeed, these cells express the chemokine receptors chemokine (C-C motif) receptor (CCR) 7, CCR2, and chemokine (C-X-C motif) receptor 4.^54,55 Monocyte chemoattractant protein 1, the chemokine for CCR2, mediates cardiac fibrosis in Ang II-induced hypertension by promoting the recruitment of BMFPCs to the heart.⁵⁶ We have measured MCP-1 in left ventricle lysates of adult WT and IL-4^−/− Balb/c mice and found a significant elevation in MCP-1 levels in WT compared with IL-4^−/− mice.¹⁰ We also detected a sizable number of CCR2-postive BMFPCs in the peripheral blood of Balb/c mice (unpublished observation, Rhaleb et al). These results suggest that IL-4-induced BMFPC homing in the heart may be MCP-1 dependent.

Role of ROS in Cardiac Fibrosis

The NADPH oxidases 2 (Nox2) and 4 (Nox4) are the 2 main Nox isoforms expressed in the heart.¹⁵ They produce ROS that participate in the differentiation of cardiac fibroblasts into myofibroblasts and play an important role in cardiac fibrosis and dysfunction.^13,57 The ROS are also known to participate in the IL-4-stimulated release and activation of cytokines such as the proinflammatory mediators, MCP-1, adhesion molecules by endothelial cells^10,58–60, and other cells such as neurons and microglia.⁶¹ However, ROS-MCP-1 signaling in the profibrotic cardiomyopathy due to high IL-4 remains unclear. We have shown that mouse cardiac fibroblasts express IL-4αR and that IL-4 increases collagen expression and production via an ROS-mediated AP-1-dependent mechanism.⁵⁷ Thus, it is possible that the increased AP-1 activity results from increased ROS, which in turn could upregulate and translocate thioredoxin and the subsequent activation of redox factor 1.⁶² Also, IL-4 increases MCP-1 secretion in cardiac fibroblasts and endothelial cells. However, it has not yet been demonstrated whether inhibition of ROS production by Nox2/Nox4 abrogates MCP-1 secretion, resulting in less BMFPC recruitment and the profibrotic effects of IL-4.

The process of how IL-4 initiates, promotes, and sustains cardiac fibrotic remodeling is unknown. The cellular source of cardiac IL-4 also remains unclear. We observed a significantly increased number of mast cells, macrophages, and CD4⁺ T cells in the myocardium of WT Balb/c compared to IL-4^−/− mice. We also found a significantly increased number of BMFPCs and an elevation in MCP-1 in the hearts of WT compared with IL-4^−/− mice. We still need to delineate whether (1) IL-4, produced by infiltrating mast cells and/or CD4⁺ T cells, activates principal cardiac cells including fibroblasts, vascular endothelial cells, and macrophages; various biological mediators released from these cells promote and progress cardiac fibrosis, making the heart more susceptible to cardiac stress and (2) IL-4-induced MCP-1 in cardiac cells mediates BMFPCs homing and cardiac fibrosis via activation of NADPH oxidase, significantly contributing to IL-4-induced fibrotic cardiomyopathy.

Role of IL-4 in Valvular Disease

Heart valvular disease could be related to autoimmune humoral and cellular response that is targeting human tissue such as the case of rheumatic heart disease (RHD). Rheumatic fever (RF) is a sequel of group A streptococcal throat infection and occurs in untreated patients. Rheumatic heart disease is a major risk factor for RF that could occur in 30% to 45% of patients with RF.⁶³ Valvular disease could be influenced by multiple factors including host’s immune response.⁶⁴ Tumor necrosis factor α (TNF)-α, interferon γ (IFN-γ), and low IL-4 levels have been observed in the rheumatic valve tissue.^64,65 These findings illustrate that exacerbated inflammatory reaction could play a vital role in the progression of valve damage. A recent article that evaluated cytokine plasma levels in patients with RHD discovered that high IL-4 and IL-10 levels predicted adverse outcome in these patients.⁶⁶ Studying circulating cytokine profile at the messenger RNA (mRNA) and protein levels could unveil potential biomarkers that could help risk stratify patients with RHD.

Role of IL-4 in Pulmonary Hypertension

On the other hand, IL-4 has been shown to induce pulmonary hypertension.^67,68 Interleukin 4 plays a role in early lung vascular inflammation, which lead to the development of pulmonary hypertension and associated right ventricular hypertrophy and fibrosis. Hypoxia-induced mitogenic factor (HIMF) plays an important role in the development of pulmonary hypertension, and it has been shown that the injection of HIMF causes less significantly endothelial cell apoptosis in IL-4 knockout (KO) mice than WT mice.⁶⁸ Also, HIMF has shown to increase the expression of IL-4 in the lungs,⁶⁷ triggering early lung inflammation by recruiting macrophages. Interestingly, HIMF induced PH and several related vascular inflammatory marker genes such as Ang2, Platelet-derived growth factor-A (PDGFA), and vascular cell adhesion molecule 1 (VCAM-1) in WT mice but not in IL-4 KO mice. Moreover, IL-4 has been shown in animal models to play a role in the development of bleomycin-induced lung fibrosis. Both IL-4- and IL-13-deficient mice showed reduced BLM-induced lung FIZZ1 expression and fibrosis and abolished in IL-4 and IL-13 double-deficient mice.⁶⁹ Hence, IL-4 could be a dependent mechanism that plays a role in the development of induced HIMF pulmonary hypertension and lung fibrosis.

Role of IL-4 in Atherosclerosis

The pathogenesis of vascular disease is complex. Many polygenic, epigenic, and inflammatory conditions trigger maladaptive pathways in response to various internal and external injuries, which results in vascular endothelial injury and then a cascade of reactions and ultimately leads to formation of atherosclerosis.⁷⁰ Interleukin 4 is present in high levels in patients with chronic inflammatory conditions and atherosclerotic lesions.⁷¹ There is growing evidence that IL-4 could also play an important role in vascular endothelial cell dysfunction and development of atherosclerosis by creating a proinflammatory vascular environment and by causing apoptosis of vascular endothelium.⁷²

Interleukin 4 has been postulated to create proinflammatory condition by 2 mechanisms. First, IL-4 causes oxidative stress-mediated upregulation of inflammatory mediators such as cytokine, chemokine, and adhesion molecules in vascular endothelial cells. Interleukin 4 itself may not cause oxidative stress and rather strengthens oxidative potential of various cells including vascular cells.⁷² Treatment of human vascular endothelial cells with IL-4 enhances the intracellular oxidizing potential as indicated by an increase in 2^′,7^′-dichlorofluorescein fluorescence.⁷³ Oxidative stress leads to disturbance of redox steady state, resulting in activation of JAK/STAT, phosphoinositide-3 kinase, and p38 mitogen-activated protein kinase signaling pathways.⁷² This causes activation and overexpression of proinflammatory mediators including IL-6, MCP-1, VCAM-1, and E-selectin. Vascular cell adhesion molecule 1 allows attachment of inflammatory cells including monocytes, macrophages, and T-cell to vascular endothelial cells, and MCP-1 facilitates internalization/transendothelial migration of these cells. Besides TNF-α and IL-1β, IL-4 upregulates and thus potentiates the effects of VCAM-1 and MCP-1.^74,75 E-selectin is also an adhesion molecule that mediates early leukocyte–endothelial interactions and is possibly controlled by IL-4 via upregulating mRNA and protein expression of E-selectin. Exact effect of IL-4 on E-selectin is still unclear.⁷² This process is called early stage of atherogenesis. This results in inflammation of intima and is associated with upregulation of scavenger receptors and Toll-like receptors (TLRs), and these receptors internalize oxidized low-density lipoprotein particles. If not sufficiently metabolized, these particles accumulate as cytoplasmic droplets in macrophages, which ultimately transform into foam cells.⁷⁰ The activated macrophages also release cytokines and metalloproteinases, which result in disruption of intima and proliferation of smooth muscle cells due to release of growth factor from macrophages and platelets. The activated Th-1 release IFN-γ establishing prothrombotic environment, and the activated CD8+ T-cells promote smooth muscle cell death and destabilizationþof plaque. All these actions augment the process of cell death, debris formation, smooth muscle cell proliferation, foam cell proliferation, and atherosclerosis.⁷⁰

Second, IL-4 regulates transcription of 15-LO-I gene in vascular endothelial cells, which may involve activation of transcription factors including STAT, AP-2, GATA motif-binding transcription factor 1, nuclear factor 1, and SP-1. This may directly create an inflammatory vascular environment or induce intracellular oxidative stress cascade leading to the atherogenesis.⁷⁶

Interleukin 4 causes accelerated apoptosis of vascular endothelial cells via activation of caspase 3 pathway, leading to dysfunction of vascular endothelial cells, and thus promotes atherosclerosis by creating an inflammatory environment.⁷²

Role of IL-4 in Macrophage Polarization

Interleukin 4 plays an important role in macrophage polarization and formation of M2 macrophage. Macrophage polarization into M1 and M2 macrophages is linked to many inflammatory and immune disorders, including aging, cardiac dysfunction, and hypertension. M1 macrophages are proinflammatory and are activated by lipopolysaccharides, INF-γ, and/or cytokines. Activated M1 macrophages release large amount of proinflammatory mediators including high-mobility group protein (HMG) B1, which transduces its signals by interacting with receptor for advanced glycation end products (RAGE), TLR2 and TLR4.⁸ Activated TLR2/TLR4 and RAGE induce nuclear factor kappaB and extracellular signal-regulated kinases 1/2 signaling, which triggers inflammatory cytokine production, such as IFNγ, IL-6, IL-1β, and TNFα, leading to inflammation and tissue destruction, including myocardial inflammation.^77–80 Similar pathogenesis is thought to be responsible for myocardial damage in aging.^77,81 This was evident in one study of macrophage polarization and cardiac inflammation in strains of accelerated senescence prone 8 (SAMP8) mice through activation of the M1 macrophage polarization and HMGB1-TLR2/TLR4 signaling pathways.⁸²

Alternatively, activated M2 macrophages have opposite role, regulation of resolution phase of inflammation, and repair of damages tissue.⁸³ M2 macrophages are stimulated by IL-4 and IL-10. Activated M2 macrophages suppress polarization of M1 macrophages, thus reduce inflammation and promote tissue repair. Interleukin 10 reduces inflammation in atherosclerosis model; M1 macrophages are found in the lipid core of human carotid atherosclerotic lesions, while M2 macrophages prevail in the shoulder region as well as in the periphery of the plaque.^84,85 Furthermore, aging has been associated with suppressed M2 macrophage polarization and increased M1 macrophage polarization leading to enhanced inflammation and myocardial dysfunction as evident in SAMP8 aging mice heart.^82,86

Pharmacological IL-4 Receptor Targets

Recombinant human IL-4 receptor was studied in patients with moderate asthma. The study showed that the drug produced significant improvement in force expiratory volume. Asthma symptom scores were stable among patients treated with IL-4 receptor (1.5 mg), despite withdrawal of corticosteroids.⁸⁷ There was no major side effect from the drug, and it is still in clinical trial. However, more experimental studies and trials are needed to further document the putative beneficial effects of neutralizing IL-4 not only on the severity of asthma symptoms but also on blood pressure control.