Roles and Mechanisms of Interleukin-12 Family Members in Cardiovascular Diseases: Opportunities and Challenges

Cardiovascular diseases represent a complex group of clinical syndromes caused by a variety of interacting pathological factors. They include the most extensive disease population and rank first in all-cause mortality worldwide. Accumulating evidence demonstrates that cytokines play critical roles in the presence and development of cardiovascular diseases. Interleukin-12 family members, including IL-12, IL-23, IL-27 and IL-35, are a class of cytokines that regulate a variety of biological effects; they are closely related to the progression of various cardiovascular diseases, including atherosclerosis, hypertension, aortic dissection, cardiac hypertrophy, myocardial infarction, and acute cardiac injury. This paper mainly discusses the role of IL-12 family members in cardiovascular diseases, and the molecular and cellular mechanisms potentially involved in their action in order to identify possible intervention targets for the prevention and clinical treatment of cardiovascular diseases.


INTRODUCTION
To date, cardiovascular disease remains the leading killer worldwide, especially in less-developed areas (Leong et al., 2017). It is not only a serious threat to patients' lives, but also poses a serious psychological burden to patients and their families. Although a large number of useful drugs and new technologies have been widely used in clinical treatment over recent years and have significantly improved survival rates, the overall prognosis of cardiovascular diseases is still very poor, and the death rate related to cardiovascular diseases is far higher than that of other diseases, even malignant tumors (Donofrio et al., 2014;Dukkipati et al., 2017;Bethel et al., 2018).

Clinical Data
Previous studies reported that plasma IL-12 concentrations are significantly increased in many types of atherosclerosis and atherosclerotic cardiovascular disease, including stable angina pectoris (SAP), non-ST segment elevation myocardial infarction (NSTEMI), ST-elevation myocardial infarction (STEMI), acute myocardial infarction (AMI), and gradually increased SAP, unstable angina pectoris (UAP), and AMI (Zhou et al., 2001;Correia et al., 2010;Lin et al., 2012;Yong et al., 2013;Chistiakov et al., 2015;Opstad et al., 2016;Zykov et al., 2016). Clinical data showed that patients with coronary artery disease exhibit higher circulating IL-23 levels (Lin et al., 2012;Abbas et al., 2015;Sun et al., 2019a). In coronary artery disease patients who underwent percutaneous coronary intervention (PCI) with drug-eluting stents (DES), subjects with in-stent restenosis show higher circulating IL-23 levels in peripheral blood mononuclear cells (PBMCs) (Khojasteh-Fard et al., 2012). Numerous studies have confirmed that IL-27 expression is increased in plasma and plaques in the coronary and carotid arteries of coronary artery disease patients (Kempe et al., 2009;Jin et al., 2012;Lin et al., 2012;A Shahi et al., 2015;Gregersen et al., 2017). Abundant evidence identifies that IL-35 expression is significantly reduced in patients with coronary artery disease; plasma IL-35 levels are gradually reduced in SAP, UAP, and AMI patients, and decreased plasma IL-35 levels are inversely correlated with the left ventricular ejection fraction (LVEF) in coronary artery diseases (Lin et al., 2012;Rasa et al., 2018;Zhu et al., 2018). Gene polymorphisms in members of the IL-12 family have been reported to be associated with the occurrence or progression of coronary artery disease. An IL-23R polymorphism was observed to be related to coronary artery disease, and the IL-23R rs6682925T/C polymorphism may independently relate to the presence of coronary artery disease (Zhang et al., 2014a). IL-27 gene polymorphism had no effect on the presence of subclinical atherosclerosis, while closely related to atherosclerosis and coronary artery disease, rs26528 T and rs40837 A alleles significantly reduced the risk of coronary artery disease (Posadas-Sánchez et al., 2017;Vargas-Alarcón et al., 2019). No research about gene polymorphisms of IL-12 and IL-35 and the presence of coronary artery disease was reported yet.

Animal Studies
Elevated serum IL-12 levels are observed of atherosclerosis in ApoE-KO mice, and increased IL-12 levels are associated with the progression of atherosclerosis (Jääskeläinen et al., 2013). Accumulating animal study reports also demonstrate that treatment with exogenous recombinant murine IL-12 significantly aggravates the progression of atherosclerosis, and increases aortic atherosclerotic plaque areas in both ApoEknockout mice and in low density lipoprotein (LDL) receptordeficient mice, while cancelation the biological effects of IL-12 can significantly diminish such effects (Lee et al., 1999;Davenport and Tipping, 2003;Hauer et al., 2005). In a murine myocardial infarction model, canceling the biological effects of IL-12 alleviates cardiac dysfunction by promoting angiogenesis (Kan et al., 2016). In a recent study, Shi et al. reported that knockout of IL-12p35 subunit, which can cancel the biological effects of IL-12 and IL-35, significantly aggravated Th1/Th2 and Th17/Treg imbalance and increased atherosclerotic plaque areas in ApoE mice, which may suggest that the pro-atherosclerotic effects of IL-12 can be mediated by promoting the CD4+ T lymphocyte differentiation imbalance .
The role of IL-23 in atherosclerosis is controversial. Therapy involving IL-23p19, a subunit of IL-23, had no significant effect on atherosclerosis development in ApoE-deficient mice, although inflammatory responses were reduced . Another study reported that there was no significant difference in atherosclerotic area between low-density lipoprotein receptor (LDLR) knockout mice and IL-23 + LDLR double-knockout mice, after they were all fed with high-fat diet (Engelbertsen et al., 2018). A recent study reported that deficiency of IL-23 significantly decreased IL-22 expression in ApoE-knockout mice, and also reduced expression of IL-22, thereby relieving the release of inflammatory substances, and thus alleviating the process of atherosclerosis (Fatkhullina et al., 2018). Subramanian et al. reported that granulocyte-macrophage colony stimulating factor (GM-CSF) up-regulates the expression of IL-23, which further promotes the differentiation of macrophages and atherosclerosis development (Subramanian et al., 2015). These studies suggest that IL-23 has a strong regulatory effect on inflammation mediated by a high-fat diet in both ApoE-knockout mice and LDL-R-knockout mice (Subramanian et al., 2015;Engelbertsen et al., 2018;Fatkhullina et al., 2018;Wang et al., 2019), while the special role of IL-23 in atherosclerotic progression is unclear and further studies are needed to clarify this aspect.
Both the effects of IL-27R and IL-27 on atherosclerosis were reported. Koltsova et al. found that knockout of IL-27R significantly enhanced Th17 immune responses, up-regulated inflammatory responses, promoted the expression of tumor necrosis factor (TNF) and IL-17A, and further promoted the development of atherosclerosis in ApoE-deficient mice (Koltsova et al., 2012). Hirase et al. also reported that knockout of IL-27 plays similar roles in atherosclerosis development in LDLRknockout mice; the mechanism may be related to the promotion of macrophage differentiation (Hirase et al., 2013). Ryu et al. found that in a high-fat diet-treated ApoE-knockout mouse atherosclerotic model, blockade of IL-27 signaling increased the plaque area via promotion of autoimmune follicular helper T cell responses (Ryu et al., 2018). These results suggest that IL-27 may be an important target for the treatment and prevention of atherosclerosis and coronary artery disease by inhibiting the differentiation of various immune cells and reducing inflammatory responses, thereby alleviating atherosclerotic progression. Hence, IL-27 may be an important target for the treatment and prevention of atherosclerosis and coronary artery disease.
Contrary to clinical experiments, as an anti-inflammatory cytokine, IL-35 expression in mouse atherosclerotic plaques was significantly increased . A small number of other studies, however, have reported increased IL-35 expression in atherosclerotic plaques and serum in ApoE mice fed with a high-fat diet, as well as in the plasma of patients with coronary artery disease (Gorzelak-Pabiś et al., 2017;Li et al., 2018). Using a mouse model of atherosclerosis, administration of recombinant mouse IL-35 significantly decreased plaque area in the aortic root, and Treg immune responses were also found to be enhanced (Tao et al., 2016). In a recent study, Shi et al. reported that knockout of IL-12p35 subunit, which can cancel the biological effects of IL-12 and IL-35, significantly aggravated Th1/Th2 and Th17/Treg imbalance and increased atherosclerotic plaque areas in ApoE mice, given that mouse IL-35 reverses Th35/Treg imbalance and up-regulates atherosclerosis development, whereas there were no effects on Th1/Th2 imbalance . In a recently published study, two subunits of IL-35 found in the left anterior descending branch following its ligation induced myocardial infarction in the heart tissues in mice. In addition, exogenous IL-35 treatment can significantly reduce infarct area of the left ventricle and reduce the incidence of left ventricular rupture; the mechanisms underlying this phenomenon may be related to the inhibitory role of IL-35 in the apoptosis of myocardial macrophages, thus increasing the differentiation of M2 macrophages and augmenting the expression of collagen (Jia et al., 2019). In rat models of coronary artery disease, IL-35 treatment significantly promotes early drug-eluting stent endothelialization; its mechanism may be related to the regulation of the activation of M2 macrophages . These studies have demonstrated that IL-35 regulates the differentiation of various immune cells involved in the progression of atherosclerotic heart disease.

Interleukin-12 Family Members and Hypertension
Hypertension is a complex group of clinical syndromes. Although the specific mechanisms remain unclear, it has been demonstrated that a variety of pathological factors are involved in the process of hypertension, among which immune responses and inflammation are most closely related to hypertension (Kirabo et al., 2014;Pober, 2014;Guzik and Touyz, 2017).
So far, little research has been conducted on IL-12 family members and hypertension. Data from clinical experiments reported that plasma IL-12 levels are significantly increased in hypertensive patients, and are positively correlated with both systolic blood pressure (SBP) and diastolic blood pressure (DBP) . IL-12 polymorphism is closely related to the incidence of hypertension-induced complications: hypertension patients who carry the IL12B 1159 A/A genotype exhibit a lower risk of incidence of stroke, while IL12B A/A carriers have an elevated risk of stroke (Timasheva et al., 2008).
In an animal study, angiotensin II (Ang II) infusion significantly increased aortic IL-12p35 expression and macrophages were the primary source . In an Ang II-induced mouse hypertension model, IL-12p35 knockout promoted M1 macrophage differentiation and elevated blood pressure, while IL-12 treatment unexpectedly lowered blood pressure . Another study reported that knockdown of IL-12p35 did not affect Ang II-induced hypertension . One possible reason for this is that the IL-12p35 knockout mice in that study were treated for only a week, which is too short a period for blood pressure to change. Little research has been conducted regarding IL-23, IL-27, and IL-35 in relation to hypertension. One group reported that in deoxycorticosterone acetate and Ang II-treated mice, deficiency of IL-17 could decrease IL-23 expression and accelerate kidney injury (Krebs et al., 2014), and another study found that treatment with recombinant mouse IL-35 had no effects on blood pressure in Ang II-treated mice .

Interleukin-12 Family Members and Aortic Aneurysms and Aortic Dissection
Aortic aneurysms and aortic dissection are both degenerative lesions of the aorta and share the same pathological mechanisms, such as the excessive loss of aortic extracellular matrix mediated by multiple pathological factors, especially local aortic inflammation (Mallat et al., 2016;Rabkin, 2017;Raffort et al., 2017;Sherifova and Holzapfel, 2019).
There have been few studies regarding IL-12 family members, aortic aneurysms, and aortic dissection. Davis et al. reported that IL-12 levels in aortic tissue and serum were not significantly different in patients with abdominal aortic aneurysms compared to those in patients without abdominal aortic aneurysms (Davis et al., 2001). In aortic dissection patients, decreased plasma IL-35 concentrations were observed compared to non-aortic dissection patients (Ye et al., 2018a). In addition, no studies have been conducted on IL-12 family members and aortic aneurysms and aortic dissection.
Only one recent animal study reported that deletion of IL-27R reduced the formation of abdominal aortic aneurysm in ApoE deficiency mice, the mechanisms may be associated with a blunted accumulation of myeloid cells in the aorta (Peshkova et al., 2019).

Interleukin-12 Family Members and Cardiac Fibrosis
Cardiac fibrosis is a common feature of many heart diseases and is closely related to deterioration in cardiac function. The essence of cardiac fibrosis is that pathological factors activate cardiac fibroblasts, leading to abnormal deposition and increased numbers of cardiac collagen fibers (Moore- Morris et al., 2015;Gorabi et al., 2019).
All studies on IL-12 family members and cardiac fibrosis have been focused on animal studies and no related clinical studies have been reported. In an earlier report, the authors reported that infusion with Ang II increases cardiac IL-12 expression derived from cardiac macrophages; detection of IL-12 promotes the activation of CD4+ T lymphocytes and increases differentiation of M2 macrophages, thereby up-regulating the activation of the transforming growth factor-b1 (TGF-b1) signaling pathway, which then aggravates cardiac fibrosis . In mouse models of myocardial infarction, deletion of IL-23 significantly reduces the expression of multiple fibrosis markers, including a-smooth muscle actin (a-SMA), collagen I, and collagen III . Unexpectedly, Yan et al. also reported that IL-23 deficiency amplifies the inflammatory response and promotes the release of various inflammatory factors, especially IL-17, which further promotes the infiltration and deposition of gdT cells in the left ventricle, promotes the apoptosis of cardiomyocytes, and aggravates cardiac fibrosis in a murine myocardial infarction model . In addition, IL-12p35 knockout increased the levels of cardiac mitochondrial reactive oxygen species (ROS) and calcium ion overload, which further aggravated mitochondrial dysfunction and energy failure, increased myocardial cell apoptosis, worsened cardiac dysfunction, and increased cardiac fibrosis in 25-month-old aging mice (Ye et al., 2020). However, how the cytokines IL-12 and IL-35 mediate these biological effects is currently unknown. Furthermore, no studies concerning IL-27 and IL-35 related to cardiac fibrosis have been reported.

Interleukin-12 Family Members and Cardiac Ischemia Reperfusion Injury
Ischemia reperfusion injury of the heart is an important issue that cannot be ignored in heart transplantation. A large number of studies have confirmed that myocardial apoptosis mediated by inflammatory responses after cardiac reperfusion is one of the most important mechanisms of ischemia reperfusion injury of the heart (Shin et al., 2017;Schanze et al., 2019).
Numerous animal studies have reported that members of the IL-12 family are involved in cardiac ischemia-reperfusion injury. In a recent study, Yan found that dectin-2 deficiency could protect against cardiac ischemia-reperfusion injury via alleviating Th1 immune responses and further decreasing IL-12 expression (Yan et al., 2017). An earlier study reported that high-mobility group box 1 (Hmgb-1) promoted ischemiareperfusion injury in a mouse cardiac transplantation model (Zhu et al., 2013). In subsequent studies, pentraxin-3 and necrostatin-1 were also found to attenuate ischemic reperfusion injury by decreasing the expression of IL-23 (Zhang et al., 2014b;Zhu et al., 2014). Hu et al. reported that administration of mouse anti-IL-23 neutralizing antibody significantly reduced the expression of inflammatory markers such as IL-6, tumor necrosis factor a (TNF-a), and pro-oxidant markers such as malondialdehyde (MDA), and decreased the levels of superoxide dismutase (SOD), thereby relieving cardiac ischemia reperfusion injury (Hu et al., 2016). Up-regulation of cardiac IL-23 expression by adenovirus significantly increased the expression of serum lactate dehydrogenase (LDH) and creatine kinase myocardial band (CK-MB), elevated the expression of apoptosis-related proteins and infarcted areas, and these effects could be reversed by AG490, an inhibitor of the JAK2-STAT pathway (Liao et al., 2017). However, the roles of both IL-27 and IL-35 in cardiac ischemia and reperfusion injury have not been studied to date.

Interleukin-12 Family Members and Atrial Fibrillation
Atrial fibrillation is one of the most common arrhythmias and can lead to vascular embolizations, the most serious of which is cerebral artery embolization. Literature reports confirm that the mechanism of atrial fibrillation may be closely related to the occurrence of atrial fibrosis (Jalife and Kaur, 2015;Nattel, 2017).
Previous clinical studies have reported that elevated IL-12 expression is observed in left atrial tissues of atrial fibrillation patients (Stein et al., 2008;Lappegård et al., 2013). Chen et al. found that IL-27 genetic variants, including the rs153109 G allele and GG genotype, increased the occurrence of atrial fibrillation in the Chinese Han population (Chen et al., 2017a).
Recent animal studies have reported that inhibition of Ang IIinduced M1 macrophage differentiation and reduction of IL-12 release can reduce the occurrence of atrial fibrosis and atrial fibrillation (Sun et al., 2019b). No studies on the expression and mechanisms of the involvement of IL-23 and IL-35 in atrial fibrillation have been reported.

Interleukin-12 Family Members and Viral Myocarditis
Viral myocarditis is an uncommon heart disease. The death rate involving severe myocarditis exceeds that of AMI. Immune responses induced by viral infection are an important cause of myocardial injury in viral myocarditis (Chen et al., 2013;Pollack et al., 2015).
So far, although no clinical experiments have been reported on IL-12 family members and viral myocarditis, a large number of animal experiments have confirmed that all IL-12 family members are associated with viral myocarditis. Substantial evidence indicates that IL-12 expression is increased in both plasma and heart tissue of coxsackievirus B3-induced viral myocarditis in mice. In addition, elevated IL-12R levels were also found in heart tissue of mice with viral myocarditis (Fairweather et al., 2003;Nyland et al., 2012;Jenke et al., 2014;Zha et al., 2015;Miteva et al., 2017;Zhang et al., 2017). In an earlier study, the authors found that treatment with coxsackievirus B3 significantly increased both cardiac IL-12p35 and IL-12p40 expression, and treatment with recombinant mouse IL-12 and anti-IL-12 neutralizing antibodies reduced and increased mortality, respectively, in mice with viral myocarditis (Shioi et al., 1997). In a subsequent study, Nishio et al. reported that carvedilol treatment increases both IL-12 and interferon-g (IFN-g) expression, thereby reducing virus replication and thus improving survival rates in viral myocarditis mice (Nishio et al., 2003). In another study, Fairweather et al. demonstrated that the protective effect of IL-12 in viral myocarditis is mediated by activation of the STAT4 pathway and promotion of IFN-g release. Knockout of the STAT4 pathway and IFN-g can significantly reverse the protective effects of IL-12 and aggravate myocardial cell injury and mortality (Fairweather et al., 2005). Similarly, circulating IL-23 levels were also observed to be increased in coxsackievirus B3induced mouse viral myocarditis (Yang et al., 2011;Sesti-Costa et al., 2017). Although there are no direct reports concerning the effects of IL-23 on viral myocarditis, emodin can reduce myocardial injury and mortality mediated by viral myocarditis by reducing the expression of IL-23, indicating that IL-23 can aggravate myocardial injury in viral myocarditis (Jiang et al., 2014). In an initial study, Kong et al. found that IL-27 levels were elevated in mice with viral myocarditis, and regulated IL-17 expression, suggesting that IL-27 may be involved in the development of viral myocarditis (Kong et al., 2014). In a follow-up study, Zhu et al. found that IL-27 inhibited immune responses to Th17 and reduced the expression of IL-17, thereby protecting against coxsackievirus B3-induced viral myocarditis (Zhu et al., 2015). Unlike other members of the IL-12 family, IL-35 levels were found to be reduced in a mouse model of viral myocarditis, and were negatively correlated with the severity of viral myocarditis, as was the frequency of Tregs (Hu et al., 2014;Ouyang et al., 2017;Xu et al., 2018). In addition, up-regulation of IL-35 expression can significantly reduce Th17-mediated immune responses, and decrease IL-17 expression, thereby alleviating cardiac injury caused by viral myocarditis (Hu et al., 2014). These studies have confirmed that all IL-12 family members are involved in the course of viral myocarditis, and the mechanisms involved in their action are related to the regulation of Th1 and Th17 immune responses. Whether other immune cells are involved needs further confirmation.

Interleukin-12 Family Members and Cardiomyopathy
Cardiomyopathy is a rare heart disease characterized by enlarged ventricular spaces with unknown etiology. Its pathological mechanisms are very complex, and many factors, including genetic variation, can induce its occurrence (Heinig et al., 2017).
Data from previous clinical experiment reported that IL-12 expression was found to be unchanged in patients with idiopathic dilated cardiomyopathy and their relatives (Marriott et al., 1996), whereas IL-12 expression was found to be elevated in patients with autoimmune cardiomyopathy or alcoholic cardiomyopathy (Izumi et al., 2000;Jenke et al., 2013;Panchenko et al., 2015). IL-12R gene polymorphisms, including IL-12B 3' UTR C and IL-12B 3' UTR CC, result in significantly higher gene expression, and may increase the incidence of Chagas cardiomyopathy (Zafra et al., 2007). Similar to the IL-12 expression trends, circulating or cardiac IL-23 levels were found to be increased in patients with dilated cardiomyopathy, and with idiopathic dilated cardiomyopathy (Yi et al., 2009;Li et al., 2010;Myers et al., 2016). Individuals with IL-12, IL-23R polymorphisms, such as SNP rs10889677, are more susceptible to dilated cardiomyopathy among the Chinese Han population, rather than those with rs1884444 and rs11465817 (Chen et al., 2009). Elevated IL-27 mRNA levels were observed in the heart tissue of human dilated cardiomyopathy patients, and an IL-27 gene polymorphism involving SNP rs153109, rather than SNP rs17855750, predisposes to dilated cardiomyopathy in the Chinese Han population (Noutsias et al., 2011;Chen et al., 2017b). The expression of IL-35 in human cardiomyopathy has not been reported.
In a mouse model of cardiac myosin immunized-mice, the absence of IL-12R significantly reduced cardiac immune responses and delayed the progression of autoimmune cardiomyopathy, whereas knockout of the STAT4 pathway and IFN-g significantly reversed the protective effect of IL-12 in autoimmune cardiomyopathy (Afanasyeva et al., 2001). In another study, Fairweather et al. found that knockout of IL-12R significantly slowed the progression of dilated cardiomyopathy in murine chronic viral myocarditis (Fairweather et al., 2004). Using IL-12p35 and IL-12p40 knockout mice and anti-IL-23 neutralizing antibodies, other researchers found that IL-23, rather than IL-12, exacerbated the progression of a localized underwear purchase response in the heart and autoimmune myocarditis, which could be blocked by anti-IL-17 neutralizing antibodies (Sonderegger et al., 2006). In a recent study, Wu et al. demonstrated that IL-23 is necessary to initiate cardiac autoimmunity by stimulating the activation and differentiation of CD4+ T lymphocytes . In contrast there have been no studies regarding cardiomyopathy and involvement of IL-27 and IL-35.

Interleukin-12 Family Members and Other Cardiovascular Diseases
IL-12 family members are also implicated in other cardiovascular diseases that are less common, such as congenital heart disease, ventricular fibrillation, and rejection after cardiac transplantation.
In earlier studies, it was reported that in young children with congenital heart disease, circulating IL-12 levels did not exhibit significant change after surgery (Madhok et al., 2006). Furthermore, the IL-27 gene polymorphism, SNP rs153109, rather than rs17855750, is associated with congenital atrial septal defects and congenital ventricular septal defects . In addition, IL-12 levels in plasma and brain tissue are significantly increased in an animal model of cardiac arrest after ventricular fibrillation. (Janata et al., 2014;Heo et al., 2017). In a mouse model of heart transplantation, administration of an anti-IL-12p40 antibody significantly reduced invasion by gdT cells, reduced the expression of various inflammatory factors, and greatly improved the survival of mice . In an animal model of acute myocardial injury induced by the chemotherapeutic drug doxorubicin, deletion of IL-12p35 significantly increased cardiac injury, which was associated with increased inflammatory responses, oxidative stress, apoptosis, and autophagy. Treatment with recombinant mouse IL-12 significantly reversed these effects, suggesting that both IL-12 and IL-35 may play protective roles in cardiac injury induced by doxorubicin (Jia, 2018;Ye et al., 2018b;Ye et al., 2018c).