*Review* **Effects of SGLT2 Inhibitors on Atherosclerosis: Lessons from Cardiovascular Clinical Outcomes in Type 2 Diabetic Patients and Basic Researches**

**Jing Xu 1,†, Taro Hirai 1,†, Daisuke Koya 1,2,3 and Munehiro Kitada 1,2,\***


**Abstract:** Atherosclerosis-caused cardiovascular diseases (CVD) are the leading cause of mortality in type 2 diabetes mellitus (T2DM). Sodium-glucose cotransporter 2 (SGLT2) inhibitors are effective oral drugs for the treatment of T2DM patients. Multiple pre-clinical and clinical studies have indicated that SGLT2 inhibitors not only reduce blood glucose but also confer benefits with regard to body weight, insulin resistance, lipid profiles and blood pressure. Recently, some cardiovascular outcome trials have demonstrated the safety and cardiovascular benefits of SGLT2 inhibitors beyond glycemic control. The SGLT2 inhibitors empagliflozin, canagliflozin, dapagliflozin and ertugliflozin reduce the rates of major adverse cardiovascular events and of hospitalization for heart failure in T2DM patients regardless of CVD. The potential mechanisms of SGLT2 inhibitors on cardioprotection may be involved in improving the function of vascular endothelial cells, suppressing oxidative stress, inhibiting inflammation and regulating autophagy, which further protect from the progression of atherosclerosis. Here, we summarized the pre-clinical and clinical evidence of SGLT2 inhibitors on cardioprotection and discussed the potential molecular mechanisms of SGLT2 inhibitors in preventing the pathogenesis of atherosclerosis and CVD.

**Keywords:** SGLT2 inhibitors; atherosclerosis; cardiovascular disease; diabetes

#### **1. Introduction**

Atherosclerosis is a chronic progressive disease characterized by the accumulation and deposition of lipids and fibrous elements in large arteries [1]. Generally, the formation of atherosclerotic plaques is divided into four stages: fatty streaks, atheromatous plaques, complicated atheromatous plaques and clinical complications [2]. Plaque rupture and thromboembolism may lead to severe cardiovascular diseases (CVD), including acute coronary syndrome (ACS), myocardial infarction (MI) or stroke [1,3]. CVD caused by atherosclerosis is the main cause of mortality in metabolic-related diseases, especially in type 2 diabetes mellitus (T2DM) [4–6]. Correspondingly, compared with non-diabetic individuals, T2DM patients have a higher risk of atherosclerosis and CVD [7,8]. Over the last decade, CVD globally affected approximately 32.2% of T2DM patients, accounting for 9.9% of deaths among them [9]. Therefore, in recent years, clinical trials of anti-diabetic agents have not only focused on hypoglycemic benefits but on cardiovascular safety and cardioprotective effects as well.

Sodium-glucose cotransporter 2 (SGLT2) is mainly localized in the proximal tubule, which is responsible for reabsorbing 80–90% of filtered glucose under physiological conditions [10,11]. The direct effect of SGLT2 inhibitors is to block glucose transportation

**Citation:** Xu, J.; Hirai, T.; Koya, D.; Kitada, M. Effects of SGLT2 Inhibitors on Atherosclerosis: Lessons from Cardiovascular Clinical Outcomes in Type 2 Diabetic Patients and Basic Researches. *J. Clin. Med.* **2022**, *11*, 137. https://doi.org/10.3390/ jcm11010137

Academic Editor: Tomoaki Ishigami

Received: 1 December 2021 Accepted: 24 December 2021 Published: 27 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in the kidney, which may depend on two distinct mechanisms: (1) by augmenting renal glucose excretion and (2) by ameliorating glucotoxicity [10]. Multiple clinical trials have demonstrated that SGLT2 inhibitors reduce blood glucose by inhibiting the reabsorption of filtered glucose in the proximal tubules independent of insulin. SGLT2 inhibitors slow the progression of macroalbuminuria, reduce the doubling of the serum creatinine level, sustain 40% reduction in the estimated glomerular filtration rate (eGFR) and further protect from inevitable renal replacement therapy or death from renal disease [12–16]. These renoprotective benefits of SGLT2 inhibitors may be attributed to glycemic control, reducing body weight, lipid profiles, blood pressure and uric acid and improving insulin resistance. Moreover, due to the improvement of these cardiovascular risk factors, SGLT2 inhibitors also present cardioprotective effects [10–19].

In this review, we summarized the clinical evidence of SGLT2 inhibitors on cardioprotection and discussed the potential molecular mechanisms of SGLT2 inhibitors in preventing the pathogenesis of atherosclerosis and CVD.

#### **2. Pathophysiological and Pharmacologic Roles of SGLT2 Inhibition**

The kidney is a crucial organ for maintaining glucose metabolism. In healthy individuals, the human kidney filters more than 180 g of glucose per day. SGLT2 is a 75 kilodalton (kDa) protein coded by the solute carrier family 5 (SLC5A2) gene in humans and is mainly localized in the kidney proximal tubule [20]. A total of 80–90% of filtered glucose is reabsorbed by SGLT2 in the early proximal tubule, while the remaining 10–20% is absorbed by SGLT1. The energy for the transport capacity of SGLT2 and SGLT1 is derived from the Na+/K+ ATPase pump located in the basolateral membrane of the proximal tubule. Therefore, the reabsorption of filtered glucose is also accompanied by Na<sup>+</sup> reabsorption [10,18,21]. SGLT2 knockout mice had glucosuria and polyuria compared with wildtype mice [22]. In patients with T2DM, the reabsorption of filtered glucose in the proximal tubules is significantly increased, contributing to elevated SGLT2 expression [23,24]. Meanwhile, the elevated reabsorption of Na+ activates the local renin-angiotensin system, stimulating the constriction of the adjacent efferent arteriole and dilation of the afferent arteriole, which leads to increases in intraglomerular pressure and glomerular filtration rate (GFR), which ultimately results in glomerular damage [10]. This evidence indicates that the inhibition of SGLT2 is a potential target for glycemic control and conferring renoprotection in treating T2DM.

The first natural SGLT inhibitor, called phlorizin, is derived from fruit trees. Phlorizin functions as a competitive inhibitor of SGLT1/2 to block glucose absorption in the proximal renal tubule and mucosa of the small intestine [25,26]. However, due to poor solubility in water, poor oral bioavailability, the non-selective inhibition of both SGLT1 and SGLT2 and a short half-life, effective studies on the mechanisms of specific SGLT2 inhibition by phlorizin are limited [25,27]. Subsequently, more novel selective SGLT2 inhibitors have been identified. Compared with phlorizin, these SGLT2 inhibitors are structurally improved. Dapagliflozin contains C-glucoside, while canagliflozin and empagliflozin contain C-glycosylated diarylmethane pharmacophore. These structures are resistant to hydrolysis by β-glucosidases to increase their half-life and specificity to SGLT2 [27,28]. To date, empagliflozin, canagliflozin, dapagliflozin and ertugliflozin have been approved by the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of T2DM [26]. Some other novel SGLT2 inhibitors, such as ipragliflozin and luseogliflozin, have been approved in Japan and other countries [29,30].

#### **3. Mechanisms of SGLT2 Inhibitors against Atherosclerosis**

During the formation of atherosclerotic plaques, three types of cells, endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and monocytes/macrophages, play crucial roles. At the first stage, due to endothelial dysfunction, excessive lipids and lipoproteins accumulate in the subendothelial matrix. This process is triggered by the accumulation of oxidized low-density lipoprotein (Ox-LDL), which stimulates the release of pro-inflammatory

cytokines, including interleukin 8 (IL-8) and adhesion molecules such as intercellular cell adhesion molecule-1 (ICAM-1), Vascular cell adhesion protein 1 (VCAM-1), monocyte chemoattractant protein 1 (MCP-1) and macrophage colony-stimulating factor (M-CSF). Then, circulating monocytes migrate to the intima, where they proliferate, differentiate into macrophages and combine with lipoproteins to form foam cells. Monocytes/macrophages express pro-inflammatory cytokines, including IL-1 and tumor necrosis factor α (TNFα). VSMCs migrating from the medial layer can also secret pro-fibrosis molecules. With the continuous migration of monocytes into the intima and the accumulation of lipids and lipoproteins, foam cells and SMCs die to form necrotic cores. Platelets will recruit to form thrombi and eventually develop atherosclerotic plaques. Vascular occlusion or plaque rupture may result in acute cardiovascular and cerebrovascular events [1,3,31].

Some metabolic characteristics, including obesity, dyslipidemia, hyperglycemia and hypertension, are independent risk factors for atherosclerosis [32]. These metabolic disorders, which may lead to endothelial dysfunction, oxidative stress, inflammation and the impairment of autophagy, participate in the formation of plaques and the pathogenesis of atherosclerosis [33]. A series of basic studies have confirmed that SGLT2 inhibitors have cardioprotective effects beyond glucose control in animal models of atherosclerosis [34–38]. The underlying mechanisms of SGLT2 inhibitors may be related to protecting endothelial function, anti-oxidative stress, anti-inflammation and maintaining basal and adaptive autophagy (Figure 1).

**Figure 1.** Mechanisms of SGLT2 inhibitors against atherosclerosis. Elevated metabolic characteristics including glucose, TC, TG, FFA, BP and angiotensin II accelerate the progression of atherosclerosis via the dysregulation of endothelial function, vasodilation and autophagy and activating oxidative stress and inflammation. SGLT2 inhibitors attenuate the alterations caused by these metabolic changes. TC, total cholesterol; TG, triglyceride; FFA, free fatty acid; BP, blood pressure; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; eNOS, endothelial nitric oxide synthase; ROS, reactive oxygen species; NF-κB, nuclear factor kappa-B; NLRP3, nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3; IL, interleukin; TNFα, tumor necrosis factor α; MCP-1, monocyte chemoattractant protein 1; ICAM-1, intercellular cell adhesion molecule-1; VCAM-1, vascular cell adhesion protein 1; AMPK, AMP-activated protein kinase; mTOR, mechanistic target of rapamycin; ↑, increase; ↓, decrease.

#### *3.1. Maintaining Endothelial Function*

Endogenous nitric oxide (NO) is a vasodilatory molecule produced by NO synthase (NOS), which presents antiatherosclerotic effects [39]. SGLT2 inhibitors inhibit cardiac Na+/K+ exchange to induce vasodilation [40]. Previous studies have shown that SGLT2 inhibitors can participate in the regulation of vasodilation by increasing the anabolism and bioavailability of NO. Both empagliflozin and canagliflozin ameliorated aortic stiffness and improved vasodilation via endothelial NOS (eNOS) phosphorylation in db/db mice and non-diabetic rats [41,42]. Empagliflozin and dapagliflozin restored NO bioavailability by suppressing reactive oxygen species (ROS) generation in TNFα-induced ECs [43].

#### *3.2. Anti-Oxidative Stress*

An imbalance between the production and scavenging of ROS results in oxidative stress, which plays crucial roles in the progression of atherosclerosis [44]. Multiple risk factors of atherosclerosis, such as high glucose, elevated free fatty acids (FFA) and triglyceride (TG), can increase ROS production via activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX1-5, p22phox, p47phox), inhibiting NOS and suppressing glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity, and then by further amplifying oxidative stress in macrophages, ECs and adipocytes and accelerating the formation of atherosclerotic plaques [45–48].

Both in vitro and in vivo experiments have confirmed the anti-oxidative stress effects of SGLT2 inhibitors. Angiotensin II can induce SGLT2 expression in ECs, which leads to the activation of oxidative stress due to NADPH oxidase. Empagliflozin presented anti-oxidant effects to protect against endothelial senescence and dysfunction via suppressing the NADPH oxidase/SGLT2 pathway [49]. Canagliflozin reduced the expression of NADPH oxidase, including NOX2, NOX4, p22phox and p47phox, and reduced the urinary excretion of 8-hydroxy-2 -deoxyguanosine (8-OHdG) in diabetic Apolipoprotein E-deficient (ApoE−/−) mice [50]. Both empagliflozin and dapagliflozin inhibited the production of ROS, ameliorated TNFα-induced oxidative stress and restored impaired NO bioavailability in human umbilical vein endothelial cells (HUVECs) and human coronary arterial endothelial cells (HCAECs) [43]. SGLT2 inhibitors, including canagliflozin, dapagliflozin and empagliflozin, protected from high-glucose-induced vasodilation disorders via suppressing NAPDH oxidase/ROS signaling in cultured ECs [51].

#### *3.3. Anti-Inflammation*

Inflammatory cytokines and inflammatory cascade reactions throughout participate in the formation, progression and rupture of atherosclerotic plaque [1,31]. Previous studies have revealed that SGLT2 inhibitors, including dapagliflozin, empagliflozin, canagliflozin and luseogliflozin, reduced a series of pro-inflammatory cytokines, including IL-1β, IL-18 [34], IL-6 and TNFα [35], and adhesion molecules such as MCP-1 [35,38,52,53], ICAM-1 [36,50] and VCAM-1 [50,52,53] in atherosclerosis animal models with or without diabetes. In vitro studies have explored the potential mechanisms of the anti-inflammatory effects of SGLT2 inhibitors. Dapagliflozin may inhibit high glucose-induced TNFα, MCP-1 and VCAM-1 via suppressing the nuclear factor kappa-B (NF-κB) pathway in human vascular endothelial cells [54]. Canagliflozin suppressed IL-1β-activated cytokine and chemokine, such as IL-6 and MCP-1, partly via AMP-activated protein kinase (AMPK) activation [55].

In addition to inflammatory pathways, inflammasome-mediated inflammatory pathways are also involved in the pathogenesis of atherosclerosis [56–58]. Ox-LDL and high glucose are responsible for the activation of the nucleotide-binding oligomerization domainlike receptor family pyrin domain containing 3 (NLRP3) inflammasome [56,59]. NLRP3 is essential for activating the precursors of IL-1β and IL-18 into their mature forms, which recruit in vascular endothelial cells, leading to atherothrombosis [58]. NF-κB promotes the transcription of NLRP3 and participates in cardiac inflammation [60], which links inflammasome and the inflammatory pathway. Moreover, TG and very low-density lipoprotein (VLDL)-related arterial inflammation are closely related to the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 1 (NLRP1) inflammasome activation in ECs [61]. Dapagliflozin inhibited IL-1β expression via NLRP3/caspase 1 signaling in streptozotocin (STZ)-induced diabetic ApoE−/<sup>−</sup> mice and T2DM rodent models [34,62,63]. Empagliflozin suppressed IL-17A-induced IL-1β and IL-18 secretions via NLRP3/caspase1 signaling and further inhibited the cell proliferation and migration of human aortic SMCs [64] and decreased the expression of IL-1β via suppressing NF-κB phosphorylation/NLRP3 signaling in human macrophages [62].

#### *3.4. Regulation of Autophagy*

Autophagy plays a complex role in the pathogenesis of atherosclerosis. Basal and mild adaptive autophagy to stress can maintain the endothelial functions of ECs, VSMCs and macrophages and protect against the formation of atherosclerotic plaques, while both deficient and excessive autophagy are related to inflammation, oxidative stress and apoptosis, which may contribute to autophagy-dependent cell death, aggravate vascular injury and lead to plaque instability or rupture [33,65,66]. Therefore, the precise regulation of autophagy is essential to prevent the development of atherosclerosis.

The SGLT2 inhibitors empagliflozin and dapagliflozin have been identified to restore autophagy deficiency in diabetic or obese rodent models, which mainly depends on the activation of nutrient-sensing pathways, such as the AMPK/mTOR (mechanistic target of rapamycin) signaling pathway [67,68]. In vitro studies have also identified several potential mechanisms. Empagliflozin restored autophagic flux impairment via activating AMPK and suppressing mTOR in H9c2 cells (rat cardiac myoblast) [69], RAW264.7 and THP-1 cells (macrophage cell lines) [70]. Our previous study also indicated that dapagliflozin had similar effects on autophagy restoration via AMPK/mTOR signaling in high-glucosetreated proximal tubular cells [71].

#### **4. Improvements of Indicators Related to CVD by SGLT2 Inhibitor**

Disorders of metabolic-related characteristics, including glucose, lipid profiles, blood pressure, uric acid, etc., contribute to increased risks of developing atherosclerosis and CVD [33]. Beyond the benefits from glycemic control, multiple clinical and rodent research studies have demonstrated that the cardiovascular benefits of SGLT2 inhibitors are closely related to the improvement of these metabolic-related characteristics.

#### *4.1. Pre-Clinical Evidence*

ApoE−/<sup>−</sup> mice and low density lipoprotein receptor knockout (Ldlr−/−) mice are well-established and extensively used rodent models of atherosclerosis [72]. Multiple studies have demonstrated that SGLT2 inhibitors, including dapagliflozin, empagliflozin, canagliflozin, luseogliflzin and ipragliflozin, reduce metabolic indicators to protect from the progression of atherosclerosis (Table 1). Dapagliflozin reduced fasting blood glucose (FBG), total cholesterol (TC) and TG [34], body weight, and glycosylated hemoglobin (HbA1c) [73] in STZ-induced diabetic ApoE−/<sup>−</sup> mice and STZ-induced diabetic Ldlr−/<sup>−</sup> mice [37]. Empagliflozin ameliorated FBG, TC, heart rate, blood pressure (BP) [53], TG, LDL [74], urinary microalbumin, body weight and fat mass [35,74] in high fat diet (HFD) fed ApoE−/<sup>−</sup> mice and reduced body weight and TG in STZ-induced diabetic ApoE−/<sup>−</sup> mice [38]. Canagliflozin decreased glucose, TC, TG, LDL and heart rate in HFD-fed diabetic ApoE−/<sup>−</sup> mice [52] and reduced TC and glucose in STZ-induced diabetic ApoE−/<sup>−</sup> mice [50]. Luseogliflozin reduced body weight, TC, TG, BP and glucose in HFD-fed mice [75] and nicotinamide in STZ-induced diabetic ApoE−/<sup>−</sup> mice [36]. Ipragliflozin decreased body weight and glucose to inhibit vascular remodeling in HFD-fed mice [76]. All these metabolic changes ameliorate vascular remodeling, reduce plaque size and increase plaque stability to protect from the progression of atherosclerosis.


#### **Table 1.** Benefits of SGLT2 inhibitors in atherosclerotic rodents.

↓, decrease; STZ, streptozotocin; Apo E, Apolipoprotein E; Ldlr, low density lipoprotein receptor; HFD, high fat diet; FBG, fasting blood glucose; TC, total cholesterol; TG, triglycerides; HbA1c, glycosylated hemoglobin; LDL, low-density lipoprotein; BP, blood pressure.

In addition to these risk factors of CVD, T2DM patients treated with SGLT2 inhibitors showed elevated plasma ketone levels [77]. SGLT2 inhibitors reduce plasma glucose concentration by increasing renal glucose excretion. To meet the energy requirements of cellules, lipid oxidation is activated, leading to the increasing of acetyl coenzyme A (CoA). Acetyl CoA can enter the Krebs cycle to be converted to ketones (acetoacetate and β-hydroxybutyrate). Moreover, enhanced lipolysis in the adipocyte leads to the increase of plasma FFA, which can also be converted to acetyl CoA via β oxidation and subsequently to ketones in the liver [10]. The elevated ketones may improve the energy metabolism of the heart. Previous studies indicated that β-hydroxybutyrateas is a strong anti-inflammatory factor. Empagliflozin can significantly increase serum β-hydroxybutyrateas to inhibit NLRP3 inflammasome and reduce the expression of IL-1β levels, which benefit from CVD [62] (Figure 2).

#### *4.2. Clinical Evidence*

SGLT2 inhibitors not only reduce high glucose independent of insulin but also improve systolic blood pressure (SBP), body weight, lipid profile and uric acid, which are considered high risk factors for CVD [10] (Figure 2). The improvement of these metabolic indicators may play a crucial role in protecting against the pathogenesis of atherosclerosis and CVD. Numerous clinical trials have confirmed that SGLT2 inhibitors can effectively reduce body weight, which may be related to increases in fat utilization, reductions in adipose tissue mass and browning in white adipose tissue, further attenuating obesity-induced insulin resistance [78–82]. Treatment with empagliflozin [83–85], canagliflozin [82,86–88] and ertugliflozin [89] reduced SBP in patients with T2DM and hypertension or patients with T2DM and chronic kidney disease (CKD). This benefit may be related to minimal natriuresis and urinary glucose excretion, which lead to weight loss, osmotic diuresis, reduced plasma volume and arterial stiffness [90]. SGLT2 inhibitors also lead to a small decrease in plasma TG, increases in high-density lipoprotein cholesterol (HDL-C) and a small increase in LDL [82,91–93]. A potential mechanism of increased LDL and decreased

TG with SGLT2 inhibition may be related to the reduction of LDL clearance, the greater lipolysis of triglyceride-rich lipoproteins [94] and the effect of switching energy metabolism from carbohydrate to lipid utilization [95]. Elevated circulating uric acid is associated with the risk of hypertension and CVD. Multiple studies have demonstrated that SGLT2 inhibitors can reduce circulating uric acid via enhancing urinary uric acid excretion in association with increased urinary glucose [96–98], which may benefit patients with CVD.

**Figure 2.** Improvements of some CVD-related clinical indicators by SGLT2 inhibitors. SGLT2 inhibitors not only reduce high glucose independent of insulin but also improve blood pressure, body weight, lipid profile and ketones and uric acid to present cardioprotective effects; ↑, increase; ↓, decrease.

#### **5. Clinical Evidence of SGLT2 Inhibitors against CVD**

Since 2008, the FDA has required that cardiovascular outcome trials (CVOTs) be carried out for new anti-diabetic drugs due to the significance of cardioprotection on T2DM patients. All potential drugs should exclude the major adverse cardiovascular events (MACE) defined in the FDA guidance, including non-fatal myocardial infarction, non-fatal stroke and cardiovascular death [99,100]. Furthermore, the effect of SGLT2 inhibitors on cardiovascular outcomes, including hospitalization for heart failure, death and CVD, compared to other glucose-lowering drugs, was also verified in a real clinical practice [101,102].

#### *5.1. Randomized Controlled Trials (RCTs)*

A meta-analysis that summarized 6 regulatory submissions and 57 published trials revealed that seven different SGLT2 inhibitors reduced the risk of Major Adverse Cardiovascular Events (MACEs) and death from any cause [103]. In this section, we summarized some landmark CVOTs of SGLT2 inhibitors (Table 2).

*J. Clin. Med.* **2022**, *11*, 137


**Table2.**Reportedcardiovascular outcometrialsofSGLT2inhibitors.

EMPA-REG OUTCOME, Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients–Removing Excess Glucose; EMPEROR-Reduced, EmpagliflozinOutcome Trial in Patients with Chronic Heart Failure with Reduced Ejection Fraction; EMPEROR-Preserved, Empagliflozin Outcome Trial in Patients with Chronic Heart Failurewith Preserved Ejection Fraction; CANVAS, Canagliflozin Cardiovascular Assessment Study; CREDENCE, Canagliflozin and Renal Events in Diabetes with Established NephropathyClinical Evaluation; DECLARE–TIMI 58, Dapagliflozin Effect on Cardiovascular Events–Thrombolysis in Myocardial Infarction 58; DAPA-HF, Dapagliflozin and Prevention of AdverseOutcomes in Heart Failure; VERTIS CV, Ertugliflozin Efficacy and Safety Cardiovascular Outcomes Trial; SOLOIST-WHF, the Effect of Sotagliflozin on Cardiovascular Events in Patientswith Type 2 Diabetes Post Worsening Heart Failure; SCORED, the Effect of Sotagliflozin on Cardiovascular and Renal Events in Patients with Type 2 Diabetes and Moderate RenalImpairment Who Are at Cardiovascular Risk; T2DM, type 2 diabetes mellitus; CKD, chronic kidney diseases; NYHA, New York Heart Association; CVD, cardiovascular diseases; EF,ejection fraction; MI, myocardial infarction. \* Primary outcomes of RCTs: EMPA-REG OUTCOME, CANVAS, CREDENCE, DECLARE–TIMI 58, VERTIS CV: MACE (the major adversecardiovascular events including a composite of death from cardiovascular causes, nonfatal myocardial infarction or nonfatal stroke); EMPEROR-Reduced, EMPEROR-Preserved: thecomposite of cardiovascular death or hospitalization for heart failure; DAPA-HF: a composite of worsening heart failure (hospitalization or an urgent visit resulting in intravenoustherapyforheartfailure)orcardiovasculardeath;SOLOIST-WHF,SCORED:thetotalnumberofdeathsfromcardiovascularcausesandhospitalizationsandurgentvisitsforheartfailure.

The Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients–Removing Excess Glucose trial (EMPA-REG OUTCOME) was the first CVOT to determine the cardiovascular benefits of SGLT2 inhibitors. This trial enrolled T2DM patients, who were treated with either empagliflozin or placebo. The primary composite outcome was MACEs. Compared with the placebo group, the empagliflozin group showed a significant reduction in death from MACEs, hospitalization for heart failure and death from any cause. However, there were no significant benefits for MI and stroke [104]. Subsequently, two trials demonstrated cardiovascular benefits in patients with heart failure and a reduced ejection fraction. The Empagliflozin Outcome Trial in Patients with Chronic Heart Failure with Reduced Ejection Fraction (EMPEROR-Reduced) enrolled 3730 patients (50% diagnosed as diabetes) with New York Heart Association (NYHA) class II to IV heart failure or with an ejection fraction (EF) ≤40%. Compared with the placebo group, the empagliflozin group had a lower risk of any inpatient or outpatient worsening heart failure event regardless of T2DM [105,106]. The Empagliflozin Outcome Trial in Patients with Chronic Heart Failure with Preserved Ejection Fraction (EMPEROR-Preserved) trial also identified the benefits of empagliflozin on heart failure in patients with NYHA class II to IV and an EF ≥40% [107].

The cardiovascular benefits of canagliflozin were demonstrated by the Canagliflozin Cardiovascular Assessment Study (CANVAS) and Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE). CANVAS involved patients with T2DM, who were treated with canagliflozin or placebo. The primary outcome was also MACEs. Similar to empagliflozin, canagliflozin showed a significant reduction in MACEs, hospitalization for heart failure and death from any cause [19]. CREDENCE confirmed the cardio and renal benefits of canagliflozin in patients with T2DM and chronic kidney disease (CKD). The primary outcome was a composite of end-stage kidney disease or death from renal or cardiovascular disease. Patients in the canagliflozin group had a lower risk of cardiovascular death, MI, stroke and hospitalization for heart failure [14].

The Dapagliflozin Effect on Cardiovascular Events–Thrombolysis in Myocardial Infarction 58 (DECLARE–TIMI 58) evaluated the cardiovascular benefits of dapagliflozin on T2DM patients. The primary outcome was MACEs. Although dapagliflozin was not found to reduce the risk of MACEs, it reduced the risk of hospitalization for heart failure [108]. Another trial, named the Dapagliflozin and Prevention of Adverse Outcomes in Heart Failure (DAPA-HF) trial, confirmed this benefit. DAPA-HF enrolled patients with heart failure and reduced ejection fraction. The primary outcome was a composite of worsening heart failure or death from cardiovascular causes. This trial also demonstrated that dapagliflozin reduced the risk of heart failure hospitalization and cardiovascular death regardless of diabetes [109].

Ertugliflozin has also completed the assessment of cardiovascular events recently. The Evaluation of Ertugliflozin Efficacy and Safety Cardiovascular Outcomes Trial (VERTIS CV) enrolled patients with T2DM and established CVD. The primary outcome was MACEs. Similar to dapagliflozin, ertugliflozin was not found to reduce the risk of MACEs, but it did reduce the risk of hospitalization for heart failure [110].

In addition to the above SGLT2 inhibitors approved by the FDA and EMA for cardiovascular indications, another SGLT2 inhibitor, sotaliflozin, also present benefits for cardiovascular death and heart failure. The Effect of Sotagliflozin on Cardiovascular Events in Patients with Type 2 Diabetes Post Worsening Heart Failure (SOLOIST-WHF) and the Effect of Sotagliflozin on Cardiovascular and Renal Events in Patients with Type 2 Diabetes and Moderate Renal Impairment Who Are at Cardiovascular Risk (SCORED) trial demonstrated that, compared with placebo, sotaliflozin treatment significantly reduced the total number of deaths from cardiovascular causes and hospitalizations and urgent visits for heart failure in T2DM patients with recent worsening heart failure [111] and T2DM patients with chronic kidney disease regardless of albuminuria [112].

According to these hallmark CVOTs, the effects of SGLT2 inhibitors on atherosclerotic cardiovascular events, such as MI and strokes, are less impressive than the effects on heart failure (Table 2). A possible reason is the heterogeneity in the CVD risk of the study populations. Besides, SGLT2 inhibitors may primarily target on ameliorating cardiac structure and function ("the pump") and not on the "pipes" (coronary arteries) [113,114]. Patients with T2DM and cardiac hypertrophy, diastolic or systolic dysfunction or with a hypervolemic state (regardless of cardiac or renal origin) are the best candidates for treatment with SGLT2 inhibitors [114].

#### *5.2. Multinational Observational Cohort Study*

The effects of drugs may be different between the RCTs and real-world practice [115,116]. Because the characteristics of patients in the RCTs do not necessarily match the standard population with T2DM, these results are difficult to be generalized.

The Comparative Effectiveness of Cardiovascular Outcomes in New Users of SGLT2 inhibitors (CVD-REAL) Study is a multinational (USA, Sweden, Norway and Denmark) observational study. In this study, 309,056 patients newly initiated on either SGLT2 inhibitors, including empagliflozin, canagliflozin and dapagliflozin, or other glucose lowering drugs were analyzed on the risk for hospitalization for heart failure and death in patients with T2DM, using clinical data in real-world practice after propensity score matching [101]. There were 961 hospitalizations for heart failure cases during 190,164 person-years followup in 6 countries, and, of 215,622 patients in the United States, Norway, Denmark, Sweden and the United Kingdom, death occurred in 1334. The use of SGLT2 inhibitors was significantly associated with lower rates of hospitalization for heart failure and death, with no significant heterogeneity by country, compared to other glucose-lowering drugs (Table 3). In addition, the sub-analysis of the CVD-REAL study exhibited an association between the initiation of SGLT2 inhibitors versus other glucose-lowering drugs and the rates of MI and stroke. Overall, 205,160 patients were included, and, in the intent-to-treat analysis, over 188,551 and 188,678 person-years of follow-up (MI and stroke, respectively), there were 1077 MI and 968 stroke events. The initiation of SGLT2 inhibitors was associated with a modestly lower risk of MI and stroke [102] (Table 3).

**Table 3.** Reported real world evidence of SGLT2 inhibitors for CVD.



**Table 3.** *Cont.*

CVD-REAL, The Comparative Effectiveness of Cardiovascular Outcomes in New Users of SGLT2 inhibitors; PAD, peripheral artery disease; MI, myocardial infarction.\* Mean follow-up: the mean follow-up time of SGLT2 inhibitors/ other glucose lowering drugs.

The CVD-REAL2 study is a similar study design to the CVD-REAL and was conducted in six countries: South Korea, Japan, Singapore, Israel, Australia and Canada [117]. After propensity score matching, the risks for death, hospitalization for heart failure, MI and stroke were analyzed in 235,064 patients of each group, newly initiated on either SGLT2 inhibitors (dapagliflozin, empagliflozin, ipragliflozin, canagliflozin, tofogliflozin and luseogliflozin) or other glucose-lowering drugs. In total, 74% of patients had no history of CVD, and patient characteristics were well-balanced in both groups. The initiation of SGLT2 inhibitors significantly reduced the risk for death, hospitalization for heart failure, MI and stroke [117] (Table 3).

In addition, a sub-analysis of the CVD-REAL-2 study showed that the initiation of SGLT2 inhibitors was associated with substantially lower risks of hospitalization for heart failure (HR 0.69, 95% CI 0.61–0.77; *p* < 0.0001), all-cause death (0.59, 0.52–0.67; *p* < 0.0001) and the composite of hospitalization for heart failure or all-cause death (0.64, 0.57–0.72; *p* < 0.0001) compared to dipeptidyl peptide-4 (DPP-4)-4 inhibitors [118]. Furthermore, in another retrospective cohort study, each of the 11,431 T2DM patients with peripheral artery disease (PAD) taking the SGLT2 inhibitor or DPP-4 inhibitor were analyzed for the risk for ischemic stroke and acute MI after propensity score matching. The use of the SGLT2 inhibitor had comparable risks of ischemic stroke and acute MI. However, the SGLT2 inhibitor group showed lower risks of congestive heart failure (HR: 0.66; 95% CI 0.49–0.89; *p* = 0.0062), lower limb ischemia requiring revascularization (HR: 0.73; 95% CI 0.54–0.98; *p* = 0.0367) or amputation (HR: 0.43; 95% CI 0.30–0.62; *p* < 0.0001) and cardiovascular death (HR: 0.67; 95% CI 0.49–0.90; *p* = 0.0089) compared to those in the DPP-4 inhibitor group. [119].

#### **6. Perspectives and Conclusions**

In recent years, as effective anti-diabetic agents, SGLT2 inhibitors have not only achieved significant results in glycemic control via increasing urinary glucose excretion independent of insulin but have also presented cardiovascular protective effects, especially in reducing the risk of MACEs and hospitalization from heart failure. Although data from the CVOTs indicated less impressive results on atherosclerotic cardiovascular events such as MI and stroke than on heart failure, some real-world practice indicated the benefits on them (Table 2). This may be related to the significant heterogeneity in the CVD risk of the study populations [113]. Clinical studies have confirmed the cardiovascular safety of SGLT2 inhibitors [19,104,108,110]. Although some common adverse events, including polyuria, genital mycotic infections, urinary tract infections and ketoacidosis, need to be carefully monitored for [19,104,108], they will not cause severe or fatal consequences. Besides, although patients need to be alerted to some other risks, such as amputation and fractures related to canagliflozin [19], there have not been significantly higher incidences than of other anti-diabetic drugs, such as glucagon-like peptide 1 receptor agonists (GLP-1RA) and

DPP-4 inhibitors [120]. Based on the safety, efficiency and cardiovascular benefits, pharmacologic recommendations from the guidelines of American Diabetes Association (ADA) recommend listing SGLT2 inhibitors for the treatment of T2DM patients with established high risk factors of atherosclerotic cardiovascular disease (ASCVD), ASCVD and heart failure [121]. The underlying mechanisms of SGLT2 inhibitors on cardioprotection may be related to improving the function of vascular endothelial cells, suppressing oxidative stress, inhibiting inflammation and regulating autophagy, which further protects from the progression of atherosclerosis. More studies are needed, however, to elucidate the underlying mechanisms.

**Author Contributions:** J.X., T.H., M.K. and D.K. contributed to drafting and writing the article. J.X., T.H., M.K. and D.K. contributed to the discussion of the review. M.K. and D.K. are responsible for the integrity of the content. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** Boehringer Ingelheim, Mitsubishi Tanabe Pharma, Taisho Pharmaceutical Co. and Ono Pharmaceutical Co. contributed to establishing the Division of Anticipatory Molecular Food Science and Technology. The authors declare that there are no conflicts of interest associated with this manuscript. The funders had no role in the interpretation or writing of the manuscript.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **References**


## *Review* **Recent Advances on the Role and Therapeutic Potential of Regulatory T Cells in Atherosclerosis**

**Toru Tanaka 1, Naoto Sasaki 1,2,\* and Yoshiyuki Rikitake <sup>1</sup>**


**Abstract:** Atherosclerotic diseases, including ischemic heart disease and stroke, are a main cause of mortality worldwide. Chronic vascular inflammation via immune dysregulation is critically involved in the pathogenesis of atherosclerosis. Accumulating evidence suggests that regulatory T cells (Tregs), responsible for maintaining immunological tolerance and suppressing excessive immune responses, play an important role in preventing the development and progression of atherosclerosis through the regulation of pathogenic immunoinflammatory responses. Several strategies to prevent and treat atherosclerosis through the promotion of regulatory immune responses have been developed, and could be clinically applied for the treatment of atherosclerotic cardiovascular disease. In this review, we summarize recent advances in our understanding of the protective role of Tregs in atherosclerosis and discuss attractive approaches to treat atherosclerotic disease by augmenting regulatory immune responses.

**Keywords:** atherosclerosis; immunology; inflammation; regulatory T cells

#### **1. Introduction**

Atherosclerosis provokes serious cardiovascular diseases (CVDs), such as ischemic heart disease and stroke, that are prominent causes of mortality worldwide. Although patients at high risk of atherosclerotic CVD usually receive state-of-the-art intensive treatment, there remains much residual risk of this disease. Experimental and clinical studies have provided firm evidence that innate and adaptive immune responses are critical factors for provoking vascular inflammation and atherosclerosis [1,2]. Although accumulating evidence suggests that vascular inflammatory responses could be responsible for the residual risk of atherosclerotic disease, we have no clinical therapies aimed to directly suppress inflammatory reactions in atherosclerotic lesions [3].

The first clinical evidence to prove the involvement of inflammation in atherosclerotic disease was provided by the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) trial, clearly showing that treatment with a fully human monoclonal antibody targeting interleukin (IL)-1β reduced the cardiovascular disease risk in patients with old myocardial infarction (MI) [4]. Other evidence has been provided by recent clinical trials, the Colchicine Cardiovascular Outcomes Trial (COLCOT) [5] and low-dose colchicine (LoDoCo2) [6], demonstrating that treatment with colchicine, an anti-inflammatory drug indicated for the treatment of gout, familial Mediterranean fever, and pericarditis, resulted in a significantly lower risk of cardiovascular events in patients with recent MI or chronic coronary disease. These clinical trials highlight the efficacy of anti-inflammatory therapies as potentially feasible strategies to treat CVD at least in patients with past disease history.

Experimental and clinical data suggest that in addition to innate immune responses, adaptive immune responses, particularly T cell-mediated immune responses, have detrimental or protective roles in atherosclerosis depending on the cell types or experimental

**Citation:** Tanaka, T.; Sasaki, N.; Rikitake, Y. Recent Advances on the Role and Therapeutic Potential of Regulatory T Cells in Atherosclerosis. *J. Clin. Med.* **2021**, *10*, 5907. https:// doi.org/10.3390/jcm10245907

Academic Editor: Tomoaki Ishigami

Received: 3 December 2021 Accepted: 13 December 2021 Published: 16 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

conditions [7]. Firm evidence indicates that regulatory T cells (Tregs), responsible for maintaining immunological tolerance and suppressing excessive immune responses [8], play an important role in preventing the development and progression of atherosclerotic disease through the regulation of pathogenic effector T cell (Teff) immune responses [9]. Several strategies to prevent and treat atherosclerosis via promoting regulatory immune responses have been developed [10]. Tuning the balance between proatherogenic Teffs and atheroprotective Tregs could be an effective therapeutic strategy for atherosclerotic disease.

In this review, we summarize recent advances in our understanding of the protective role of Tregs in atherosclerosis, and discuss attractive approaches to treat atherosclerotic disease by augmenting regulatory immune responses.

#### **2. Role of the Immune System in Atherosclerotic Disease**

Immune dysregulation causes chronic inflammation in the arterial wall, which is a key feature of atherosclerosis [11] (Figure 1). It has been suggested that the dysregulated arterial immunoinflammatory responses could induce atherosclerotic plaque instability and eventually lead to severe clinical events, including acute coronary syndrome (ACS) and stroke. It is believed that as the initial inflammatory response in atherosclerosis, accumulation and retention of low-density lipoprotein (LDL) in the arterial intima could occur. Upon endothelial activation, inflammatory monocytes enter the subendothelial space or the intima of the arterial wall, differentiate into macrophages, and form lipid-laden foam cells via uptake of modified LDL particles, leading to the promotion of arterial immunoinflammatory responses including proatherogenic Teff immune responses. A large number of experimental and clinical studies have revealed that various immune cells that mediate innate and adaptive immunity are deeply involved in the pathogenesis of atherosclerosis [1]. In particular, genetically altered mouse models of atherosclerosis including the apolipoprotein E-deficient (*Apoe*−/−) [12] and low-density lipoprotein receptor-deficient (*Ldlr*−/−) mice [13] have substantially contributed to the development of the atherosclerosis research field.

**Figure 1.** Role of the immune system in atherosclerosis. Chronic vascular inflammation via immune dysregulation is critically involved in the pathogenesis of atherogenesis. Regulatory T cells (Tregs) protect against atherosclerosis by suppressing activation of various immune cells. DC, dendritic cell; IL, interleukin; LDL, low-density lipoprotein; Teff, effector T cell; TGF, transforming growth factor.

Naïve T cells are presented with some atherosclerosis-associated antigens by antigenpresenting cells (APCs), and differentiate into activated Teffs, including CD4+ or CD8<sup>+</sup> T cells that accumulate into atherosclerotic lesions of atherosclerosis-prone *Apoe*−/<sup>−</sup> or *Ldlr*−/<sup>−</sup> mice and humans. Dendritic cells (DCs) have a capacity to present antigens and play complex roles in atherosclerosis. Various subsets of DCs have been shown to play protective or detrimental roles in atherosclerosis, depending on their subsets [14]. The finding of oligoclonal expansion of T cells in mouse atherosclerotic lesions indicates that T cells may expand in an antigen-specific manner within atherosclerotic plaques [15]. Several self-antigens, native LDL, oxidized LDL, and the major component of LDL, such as apolipoprotein B (ApoB), could provoke autoimmune responses in atherosclerotic lesions and be involved in the pathogenesis of atherosclerosis [16]. However, the role of these atherosclerosis-specific antigens in the development of atherosclerosis remains obscure, and a detailed analysis is needed. With the help of T cells and antigenic stimulation, B cell differentiation and activation occur. Recent experimental studies have revealed that several subsets of B cells play differential and complex roles in atherosclerosis [17]. Conventional B2 cells contribute to proatherogenic immune responses, while B1 cells or regulatory B cells protect against atherosclerosis by secreting natural IgM antibodies against oxidation-specific epitopes or IL-10, respectively.

Depending on the expression of specific transcriptional factors or variations in the local cytokine milieu along with antigen presentation, naïve CD4+ T cells differentiate into different Teff lineages, including T helper type 1 (Th1), T helper type 2 (Th2), and T helper type 17 (Th17) lineages, which have critical roles in the development of atherosclerosis in mice and humans [1]. Recent experimental and clinical studies using sophisticated methodologies, such as single-cell RNA sequencing and mass cytometry revealed that a distinct subset of T cells is the major cellular component in atherosclerotic plaques of mice [18] and humans [19]. Th1 cells specifically express the transcription factor T-box expressed in T cells (T-bet) and secrete pro-inflammatory cytokines, interferon (IFN)-γ, IL-2, and tumor necrosis factor (TNF)-α. Among these cytokines, the major Th1 cytokine, IFN-γ, has been shown to have potent pro-atherogenic effects [20,21]. Th1 cells predominantly exist in mouse [22] and human [23] atherosclerotic plaques and promote atherosclerosis. Th2 cells specifically expressing the transcription factor GATA3 secrete IL-4, IL-5, IL-10, and IL-13, and its major cytokine is IL-4. The genetic deletion of IL-4 in *Ldlr*−/<sup>−</sup> mice reduces atherosclerosis [24], while exogenous administration of IL-4 did not affect atherosclerosis in *Apoe*−/<sup>−</sup> mice [25], producing discrepant results. The role of Th2-mediated immune responses in atherosclerosis is heavily influenced by the secreted specific cytokines or animal models and remains controversial. Th17 cells specifically expressing the transcription factor RAR-related orphan receptor (ROR)-γt produce pro-inflammatory cytokine IL-17, and have been shown to substantially contribute to the development of several autoimmune diseases [26]. However, pharmacological inhibition of IL-17 function or its genetic inactivation yielded discrepant outcomes in atherosclerosis [27–29]. The role of Th17 cells in atherosclerosis may vary depending on animal models or experimental conditions, and further investigations are required. CD8+ T cells also infiltrate into atherosclerotic lesions, and their numbers are higher than CD4<sup>+</sup> T cells in advanced human atherosclerotic lesions [19]. Experiments performing CD8α or CD8β monoclonal antibody-mediated depletion of CD8<sup>+</sup> T cells in *Apoe*−/<sup>−</sup> mice or their transfer into lymphocyte-deficient *Apoe*−/<sup>−</sup> mice revealed that CD8<sup>+</sup> T cells promote the development of atherosclerotic lesions with an unstable plaque phenotype [30]. On the other hand, depletion of CD8<sup>+</sup> T cells by injecting CD8α-depleting antibodies in *Ldlr*−/<sup>−</sup> mice resulted in less stable plaques characterized by reduced collagen content and increased macrophage content and necrosis, though that did not affect atherosclerotic lesion size [31]. Thus, CD8<sup>+</sup> T cells have pro- and anti-atherogenic actions depending on experimental models.

T cell activation occurs after receiving two signals from APCs that involve interaction of the T cell receptor (TCR) with antigenic peptide/major histocompatibility complex (MHC) ligand and costimulation. The costimulatory signals promote or inhibit the

TCR-MHC signal, depending on the type of costimulation. The T cell costimulatory and coinhibitory pathways play crucial roles in modulating functions of Teffs and Tregs and their balance, and these pathways have been shown to be involved in the pathogenesis of atherosclerosis in recent animal studies using genetically modified mice or neutralizing antibodies [32]. Activated T cells and forkhead box P3 (Foxp3)<sup>+</sup> Tregs highly express the coinhibitory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds to B7 family molecules, B7-1 (CD80) and B7-2 (CD86), on APCs and interrupts the interaction of these molecules with CD28 on T cells, resulting in potent suppression of T cell activation [33]. Our recent studies using CTLA-4 transgenic mice on an *Apoe*−/<sup>−</sup> background in which T cells constitutively express CTLA-4 demonstrated that CTLA-4 overexpression protected against the development of atherosclerosis [34], abdominal aortic aneurysm (AAA) [35], and kidney disease [36] by suppressing maturation of DCs and proinflammatory Teff immune responses. CTLA-4-Ig that binds to CD80 and CD86 and has an inhibitory effect on the CD80/CD86–CD28 costimulation pathway is clinically effective in treating rheumatoid arthritis [37], and has also been shown to protect against experimental atherosclerosis [38]. These reports suggest that CTLA-4 could be a possible therapeutic target for atherosclerosis. Another important coinhibitory pathway is the interactions between programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1)/PD-L2. Genetic deletion of both PD-L1 and PD-L2 [39] or of their receptor PD-1 [40] in *Ldlr*−/<sup>−</sup> mice led to accelerated development of atherosclerotic lesions with increased accumulation of intraplaque CD4+ and CD8<sup>+</sup> T cells, indicating an atheroprotective role of the PD-1–PD-L1/PD-L2 pathway.

In recent years, the new field termed immuno-oncology has emerged, and immunotherapies for various types of cancer have attained great success. Monoclonal antibodies to block PD-1, PD-L1, or CTLA-4, called immune checkpoint inhibitors (ICIs), have been developed. Recent experimental and clinical evidence suggests that stimulation of the antitumor functions of T cells with these ICIs is quite effective for the treatment of cancer, and has become a major treatment for advanced unresectable cancer [41]. In consideration of findings obtained from animal studies showing that these inhibitory molecules are negative regulators of atherosclerosis [34,38–40], treatment with ICIs have the potential to accelerate atherosclerosis in cancer patients who could have a higher risk of CVD. Importantly, a recent clinical trial has demonstrated that treatment with ICIs was associated with a higher risk of atherosclerosis-related cardiovascular events in patients with various types of cancer, providing evidence that T cell activation is critically involved in the development of atherosclerotic disease in humans [42]. Careful management is required for the use of ICIs for treating cancer patients with cardiovascular risk factors.

#### **3. Protective Role of Tregs in Atherosclerotic Disease**

Thymus-derived natural Tregs were discovered by Sakaguchi et al. in 1995 [43]. Tregs constitutively express high levels of CD25 (IL-2 receptor α-chain) molecule [43] and the transcription factor Foxp3, an essential factor for their differentiation and function [44,45]. Impaired Treg function has been shown to be a primary cause of several autoimmune diseases in mice [46] and humans [47], indicating that this cell population plays an indispensable role in the dominant suppression of autoimmune responses and the maintenance of immune homeostasis. Tregs have multiple inhibitory actions, including suppression of autoimmune T cell proliferation and its differentiation into Th1, Th2, and Th17 lineage, and the inactivation of various immune cells, including B cells, DCs, and macrophages [48]. Several lines of evidence indicate that Tregs protect against atherosclerosis by regulating pathogenic immunoinflammatory responses (Figure 1 and Table 1). Foxp3+ Tregs could also be generated from naïve T cells in the periphery, such as gut-associated lymphoid tissue (GALT), which are called peripherally derived Tregs (pTregs) that maintain mucosal immune tolerance and suppress autoimmune responses [49]. However, the differences in their characteristics between thymus-derived natural Tregs and pTregs remain obscure, and further investigation will be needed. For more detailed information about the diverse role of Tregs in immunology, the reader is referred to a recent review [8].

#### *3.1. Protective Role of Tregs in Experimental Atherosclerosis*

The costimulatory pathway CD80/CD86–CD28 plays a role in the generation and homeostasis of Tregs [50]. Genetic deficiency of this costimulatory signaling exacerbated atherosclerosis in *Ldlr*−/<sup>−</sup> or *Apoe*−/<sup>−</sup> mice, and was associated with a significant decrease in CD4+CD25+ Treg numbers in lymphoid tissues [51]. Notably, adoptive transfer of CD4+CD25+ Tregs abrogated the detrimental effects on atherosclerosis observed in *Apoe*−/<sup>−</sup> mice with the CD80/CD86–CD28 pathway deficiency [51]. In line with this, another research group reported that adoptive transfer of CD4+CD25+ Tregs attenuated the development of atherosclerosis in Treg-competent *Apoe*−/<sup>−</sup> mice [52]. These findings provided the novel concept that CD4+CD25+ Tregs protect against atherosclerosis in mice under hypercholesterolemia. A recent study investigated the role of another costimulatory pathway, CD27–CD70, in atherosclerosis by inducing bone marrow-derived and systemic CD27 deficiency in *Apoe*−/<sup>−</sup> mice, and showed that CD27 deficiency exacerbated early stages of atherosclerosis, along with reduced Treg numbers in various lymphoid organs and the aorta [53]. Interestingly, this study also demonstrated that adoptive transfer of wild-type CD4+CD25+ Tregs expressing folate receptor 4 into CD27-deficient *Apoe*−/<sup>−</sup> mice reversed the phenotype of atherosclerosis, indicating a causative role of decreased Treg frequency in CD27-deficiency-dependent atherosclerosis progression [53].

An important issue for the definition of Tregs is that CD25-expressing CD4<sup>+</sup> T cell population may contain activated conventional T cells. Firm experimental evidence supports that Foxp3 is the most reliable molecular marker for Tregs in mice [48]. To investigate the precise role of Tregs in atherosclerosis, Klingenberg et al. utilized the DEREG (depletion of regulatory T cells) mice, in which Foxp3+ Tregs faithfully express a diphtheria toxin receptor and can be specifically depleted by diphtheria toxin administration [54]. Treg deficiency by transplanting DEREG bone marrow into lethally irradiated *Ldlr*−/<sup>−</sup> mice accelerated atherosclerosis development without affecting aortic inflammatory responses [55]. This pro-atherogenic effect caused by Treg depletion was associated with markedly elevated plasma cholesterol levels in the very low-density lipoprotein and chylomicron remnant fractions [55]. This study identified for the first time the role of Foxp3<sup>+</sup> Tregs in atherosclerosis and demonstrated a novel suppressive mechanism that modulates lipid metabolism other than the well-recognized inflammation regulation.

#### *3.2. Possible Protective Role of Tregs in Human Atherosclerosis*

In addition to the strong experimental evidence for atheroprotective actions of Tregs, accumulating evidence highlights their importance in human atherosclerotic disease. Low numbers of FOXP3+ Tregs were detected in all the progression stages of human atherosclerotic plaques [56]. Single-cell RNA sequencing of a broad cohort of human carotid plaques identified a small sized cluster, showing Tregs detected by the expression of *FOXP3*, *CD25*, and *CTLA4* [57]. Although the number and phenotype of Tregs in human atherosclerotic lesions have not been extensively explored, there are a number of studies that examined the correlation between circulating Treg levels and coronary artery disease (CAD) [58–62], showing reduced peripheral Treg numbers in ACS patients compared with healthy controls or stable angina patients [58,61]. A recent large and long follow-up study showed an association between low levels of baseline CD4+FOXP3+ Tregs and an increased risk of acute coronary events, but not stroke [63]. The CD4+CD25+CD127low Treg counts in coronary thrombi were significantly increased compared with peripheral blood in patients with ST elevation or non-ST elevation ACS, indicating that peripheral Tregs might decrease due to their accumulation in ACS lesions in the acute phase of MI [64]. Together, these results suggest that decreased numbers of Tregs may be responsible for the pathogenic inflammation in ACS.

Several markers, such as CD25, CD127, and FOXP3 have been used to define Tregs in previous clinical studies. The strategy of staining the FOXP3 molecule can discriminate Tregs from Teffs more precisely than by the combination of CD25 and CD127 molecules. However, as opposed to murine CD4+Foxp3+ T cells, FOXP3 expression is induced in human CD4+FOXP3<sup>−</sup> T cells upon TCR stimulation, which do not exhibit suppressive functions, and therefore such a population may contain some Teffs [8]. Miyara et al. proposed a new strategy to define human Tregs, demonstrating that Tregs are separated into two subsets (CD4+CD45RA+FOXP3low resting Tregs and CD4+CD45RA−FOXP3high activated Tregs) and that activated Teffs are defined as CD4+CD45RA−FOXP3low T cells or CD4+CD45RA<sup>−</sup>FOXP3<sup>−</sup> T cells [65]. The combination of CD45RA and FOXP3 staining of CD4<sup>+</sup> T cells may identify Tregs in human peripheral blood more precisely than previous methods. Using this strategy to accurately define human Tregs, we reported that both resting Treg and activated Treg levels were decreased, whereas the CD4+CD45RA<sup>−</sup>FOXP3<sup>−</sup> fraction of activated Teffs was increased in the peripheral blood of patients with stable angina pectoris and old MI compared with healthy controls [66]. These results imply that dysregulated Treg responses might promote atherosclerosis in humans, though it remains unclear whether the imbalance between proatherogenic Teffs and atheroprotective Tregs is a cause or result of CAD. Another important issue regarding the difference in FOXP3 expression between mice and humans is that there are several different isoforms for FOXP3, including FOXP3 lacking the region encoded by exon 2 (FOXP3Δ2). A recent clinical study revealed that not total FOXP3 levels but the proportion of FOXP3Δ2 expression was associated with symptomatic atherosclerotic disease, and that Treg activation led to the induction of FOXP3Δ2 isoform expression that plays an important role in the regulation of its effector functions [67]. Collectively, these reports highlight the possible role of Tregs in the protection against human atherosclerotic disease.

#### *3.3. Protective Role of Tregs in AAA*

AAA characterized by dilation of the abdominal aorta is a lethal aortic disease and associated with atherosclerosis. Importantly, there are no effective pharmacological therapies against this disease, and surgical treatment is performed if the risk of rupture is higher than that of the procedure. Therefore, it is important to elucidate the detailed mechanisms underlying AAA development and develop noninvasive therapies for this disease. Clinical and experimental evidence suggests that inflammation caused by accumulation of Teffs and macrophages in aneurysmal lesions contributes to the development of AAA, and that Tregs protect against AAA formation [68]. Genetic deficiency of CD4+CD25+ Tregs promotes the development and rupture of angiotensin II-induced AAA in normocholesterolemic mice [69]. Adoptive transfer of CD4+CD25+ Tregs prevents angiotensin II-induced AAA formation in atherosclerosis-prone *Apoe*−/<sup>−</sup> mice [70]. Using hypercholesterolemic DEREG mice, we demonstrated that genetic depletion of CD4+Foxp3+ Tregs aggravates AAA formation and rupture in angiotensin II-infused *Apoe*−/<sup>−</sup> mice by upregulating immunoinflammatory responses in the aneurysmal lesions, providing direct evidence for a protective role of CD4+Foxp3+ Tregs in the development of AAA [71]. In patients with AAA, decreased numbers of CD4+CD25+FOXP3+ Tregs were observed, and Foxp3 expression in peripheral CD4+CD25+ cells was decreased compared with healthy control subjects [72]. FOXP3 expression levels in human aneurysmal tissues are significantly lower than in normal thoracic aortic tissues [70]. These reports suggest that impaired immunoregulation by Tregs may be involved in the development of AAA, and that promotion of endogenous regulatory immune responses may represent an attractive therapeutic approach to AAA.

#### *3.4. Mechanisms of Treg-Mediated Atheroprotection*

A large number of studies in immunology fields have elucidated the mechanisms by which Tregs control pathogenic immune responses, leading to the development of effective approaches to prevent and treat inflammatory diseases based on the modulation of their functions. Tregs exert suppressive functions via multiple mechanisms, including

cell contact-dependent suppression, secretion of immunosuppressive factors including IL-10, IL-35, and transforming growth factor (TGF)-β, intracellular molecule (granzyme, cyclic adenosine monophosphate, and indoleamine 2,3-dioxygenase)-dependent suppression, and IL-2 deprivation from responder T cells, which could operate in a synergistical or complementary manner [8]. Although it remains obscure which mechanism is dominant for the Treg-mediated regulation of pathogenic immune responses, core suppressive pathways may depend on the type and stage of disease, or subsets of Tregs.

Among cytokines secreted from T cells, IL-10 and TGF-β have potent anti-inflammatory actions and have attracted much attention as potent anti-atherosclerotic cytokines [73]. Regarding the suppressive actions of Tregs in atherosclerosis, IL-10 and TGF-β production might be involved in this process. In *Ldlr*−/<sup>−</sup> mice fed a high-cholesterol diet, overexpression of IL-10 in T cells inhibited the development of advanced atherosclerotic lesions by shifting the Th1/Th2 balance toward Th2 phenotype [74]. Genetic deletion of TGF-β signaling in T cells led to a dramatic increase in atherosclerotic lesion development with an unstable plaque phenotype in *Apoe*−/<sup>−</sup> mice [75]. As described above, our recent study demonstrated that overexpression of CTLA-4 in T cells inhibited atherosclerosis development in *Apoe*−/<sup>−</sup> mice by downregulating the CD80 and CD86 expression on DCs and limiting the CD80/CD86–CD28-dependent activation of Teffs [34]. CTLA-4-dependent suppression of DC function by Tregs may also be involved in the reduction in atherosclerosis. However, it remains to be determined whether these suppressive mechanisms depend on Tregs or other subsets of T cells. Tregs contribute to the promotion of inflammation resolution by enhancing apoptotic cell clearance (efferocytosis) by macrophages, which could contribute to the prevention of atherosclerosis development [76]. The identification of dominant atheroprotective mechanisms mediated by Tregs would lead to the establishment of novel Treg-based therapies for atherosclerotic disease.

#### *3.5. Treg Immune Responses under Hypercholesterolemia*

Several experimental reports have shown the close link between hypercholesterolemia and Treg number and function (Table 1). The proportion of Foxp3+ Tregs within the splenic CD4<sup>+</sup> T cell population was markedly increased in *Ldlr*−/<sup>−</sup> mice fed a cholesterol-rich diet, whereas their accumulation in atherosclerotic plaques was impaired in association with decreased expression of their surface molecules related to migratory function, which was prevented by reversal of hypercholesterolemia [77]. The in vitro suppressive function of Tregs was maintained under hypercholesterolemia, although their expression of various selectin ligands and binding capacity to aortic endothelium were decreased and apoptosis of intraplaque Tregs was promoted under such conditions [77]. Another experimental study in wild-type mice fed a cholesterol-rich diet demonstrated that diet-induced hypercholesterolemia rather increased an in vitro Treg suppressive activity [78]. In *Ldlr*–/– mice fed a high-cholesterol diet exhibiting mild hypercholesterolemia, Treg differentiation was promoted in the liver [79]. However, under severe hypercholesterolemia, disrupted homeostasis in the liver promoted Th1 cell differentiation and CD11b+CD11c+ leukocyte accumulation, resulting in the abrogation of Treg responses and the promotion of atherosclerosis [79]. A recent study in *Ldlr*−/<sup>−</sup> mice fed a high-cholesterol diet has shown that diet-induced hypercholesterolemia modulated the intracellular metabolism of Tregs and the expression of several molecules involved in their migration, which led to the conversion of Tregs to an effector-like migratory phenotype and the promotion of their migration towards atherosclerotic aortas [80]. These results imply that decreased Treg migratory capacity due to hypercholesterolemia may not be responsible for the dysfunction of their ability to control atherosclerosis. Thus, accumulating experimental evidence indicates that hypercholesterolemia affects the number, function, migratory capacity, and intracellular metabolism in Tregs. Further studies are needed to better understand these mechanisms.

It is supposed that Tregs in circulation enter the lymphoid tissues surrounding atherosclerotic arteries where they may be presented with some atherosclerosis-associated antigens by DCs, become activated, and migrate into atherosclerotic aortas to mitigate

lesional inflammation and plaque development. This implies that the expansion of Tregs in atherosclerotic lesions could lead to reduced plaque inflammation and lesion development. The chemokine system plays an important role in the recruitment of T cells and monocytes to atherosclerotic lesions [81]. Several chemokine receptors are specifically expressed on each Th subset, whereas Treg-specific chemokine receptors have not been identified. An experimental report demonstrated that the expression of the chemokine CX3CL1 was selectively upregulated in the aorta of *Ldlr*−/<sup>−</sup> mice fed a high-cholesterol diet compared with other lymphoid tissues, and that the adoptive transfer of its counterreceptor CX3CR1 transduced Tregs promoted their migration to the atherosclerotic lesions and reduced atherosclerosis development, although CX3CR1 is mainly expressed on Ly6C+ monocytes and its endogenous expression on Tregs is relatively low [82]. The role of chemokine– chemokine receptor interactions in Treg recruitment to atherosclerotic lesions remains largely unknown. Importantly, it remains unclear whether lesional Tregs suppress inflammatory reactions responsible for atherosclerotic lesion development, and further extensive experiments are required to identify the exact role of lesional Tregs in atherosclerosis.

#### *3.6. Stability, Plasticity, and Antigen-Specificity of Tregs in Atherosclerosis*

Tregs may stably exert suppressive activities for a long time under physiological conditions, while they have been reported to lose inhibitory functions (instability) and show an effector-like phenotype (plasticity) under inflammatory conditions (Table 1) [83]. Similar phenomena are observed under the conditions of prolonged hypercholesterolemia [84]. In *Apoe*−/<sup>−</sup> mice, CD4+Foxp3+ Tregs could differentiate into proinflammatory Th1-like cells in the aorta and secondary lymphoid tissues and become dysfunctional, leading to the exacerbation of arterial inflammation and lesion development [85]. CD4+Foxp3+CCR5+CD25<sup>−</sup> Tregs found exclusively in the aorta and draining lymph nodes of *Apoe*−/<sup>−</sup> mice fed a highcholesterol diet for a long period exhibit impaired suppressive activities and exacerbate atherosclerosis, although it remains unclear whether this cell population is derived from bona fide Tregs [86].

**Table 1.** Role of Tregs in atherosclerotic disease.




*Apoe*−/−, apolipoprotein E-deficient; CAD, coronary artery disease; CTLA-4, cytotoxic T-lymphocyte-associated antigen-4; DC, dendritic cell; Foxp3, forkhead box P3; IL, interleukin; *Ldlr*−/−, low-density lipoprotein receptordeficient; TGF, transforming growth factor; Th1, T helper type 1; Treg, regulatory T cell.

As the disruption of immunologic tolerance against modified lipoproteins may be responsible for the pathogenesis of atherosclerosis, it could be considered as an autoimmune disease. T cells including Tregs respond to some antigens derived from components of LDL particles [7]. MHC class II-deficient *Apoe*−/<sup>−</sup> mice had greater atherosclerotic lesions and reduced numbers of Tregs, implying that MHC class II-mediated generation and activation of antigen-specific Tregs would have a protective role in the development of atherosclerosis [87]. However, due to technical limitations, the role of antigen-specific T cells in atherosclerosis has not been elucidated in most experimental or clinical studies. A recent study using newly designed tetramers to detect human T cells specific for ApoB-derived peptides ApoB3036–3050, possible atherosclerosis-related antigens, has shown that healthy subjects have ApoB-specific Foxp3<sup>+</sup> Tregs, and that these Tregs coexpress other CD4 lineage transcription factors, such as ROR-γt, in patients with subclinical cardiovascular disease [88]. Another study by the same group using a tetramer to detect ApoB978–993-specific T cells has revealed that ApoB-specific T cells in *Apoe*−/<sup>−</sup> mice convert from mixed Th17/Treg cells with a regulatory anti-inflammatory transcriptome into proinflammatory Th1/Th17-like cells that secrete inflammatory cytokines, and that these ApoB-specific CD4+ T cells with a predominant Th1/Th17 phenotype were detected in the blood of patients with CAD [89]. Many questions remain to be answered regarding the role of stability, plasticity, and antigen specificity of Tregs in atherosclerosis [7]. Further studies should address these issues to translate findings obtained by experimental studies to the clinical settings.

#### **4. Therapeutic Approaches for Atherosclerotic Disease by Promoting Regulatory Immune Responses**

The balance between Teffs and Tregs is critical for the control of atherosclerotic disease. Based on recent experimental and clinical evidence, we expect that improving this balance, by enhancing Treg responses or dampening Teff responses, could be an effective therapy for atherosclerotic disease [9,10] (Figure 2). A large number of experimental studies have shown that the activation and expansion of antigen-specific or non-specific Tregs protect against atherosclerosis by suppressing pathogenic immune responses (Table 2). This may be supported by the fact that upon antigen presentation, Tregs also exert suppressive functions in an antigen-non-specific manner [48].

**Figure 2.** The balance between proatherogenic effector T cells (Teffs) and atheroprotective regulatory T cells (Tregs) is critical for the control of atherosclerosis.

#### *4.1. Vaccination Strategies*

Experimental studies in atherosclerosis-prone mice have demonstrated that immunization with several atherosclerosis-related antigens, native LDL, oxidized LDL, or ApoBderived peptides, induces antigen-specific Tregs and attenuates atherosclerotic lesion development [16]. The induction of antigen-specific immunological tolerance could be an attractive therapeutic strategy for atherosclerosis, because this approach can dampen pathogenic immune responses against atherosclerosis-related antigens and avoid general immunosuppression. The identification of atherosclerosis-related antigens and the development of effective immunization methods would advance the field in designing a novel approach to prevent human atherosclerosis. For more in-depth information on vaccination strategies in atherosclerosis, the reader is referred to a recent review [16].

#### *4.2. Modulation of DC Functions*

The interaction between Tregs and some DC subsets play a protective role in atherosclerosis [9]. DC maturation mediated by MyD88, a key Toll-like receptor adaptor, is atheroprotective through controlling Treg responses in Western-type diet-fed *Ldlr*−/<sup>−</sup> mice [90]. Flt3 signaling-dependent CD11chiMHC-IIhiCD11b−CD103+ DCs in aortic tissue promote accumulation of CD4+Foxp3+ Tregs and protect against atherosclerosis in *Ldlr*−/<sup>−</sup> mice fed a high-fat diet [91]. Immature tolerogenic DCs, characterized by low expression levels of MHC class II molecule and costimulatory molecules, expand CD4+Foxp3+ Tregs and inhibit Teff responses, contributing to the maintenance of immune tolerance and regulation of atherosclerosis [92]. Adoptive transfer of ApoB100-pulsed tolerogenic DCs prevented atherosclerosis development in hypercholesterolemic mice by diminishing pathogenic T cell responses to ApoB100 and promoting antigen-specific CD4+Foxp3+ Treg responses [93]. Our study showed that oral administration of the active form of vitamin D3 (calcitriol) attenuated atherosclerosis development in *Apoe*−/<sup>−</sup> mice by expanding tolerogenic DCs and CD4+Foxp3+ Tregs systemically and locally in atherosclerotic lesions, which might interact

with each other and efficiently regulate aortic inflammatory responses associated with atherosclerosis [94]. This study provided direct evidence for a possible anti-atherogenic role of vitamin D reported in an epidemiological study showing the association between vitamin D deficiency and increased cardiovascular events and mortality [95]. Collectively, strategies to expand and activate several types of atheroprotective DCs could be possible therapies for the prevention of atherosclerosis [92].

#### *4.3. Modulation of Intestinal Immunity*

As described above, Foxp3<sup>+</sup> Tregs (known as pTregs) differentiate from naïve T cells not only in the thymus but also in GALT, where multiple stimuli such as commensal microbiota and food antigens could effectively promote their induction [49,96]. T regulatory type 1 (Tr1) or T helper type 3 (Th3) cells that do not express Foxp3 are induced in GALT and produce immunomodulatory cytokines IL-10 or TGF-β, respectively [96]. The development of atherosclerosis was attenuated by in vivo induction of Tr1 cells in atherosclerosis-prone mice, which was associated with increased production of IL-10 [97,98]. Experimental evidence indicates that induction of mucosal (oral or nasal) tolerance may be an effective way to prevent and treat various autoimmune diseases, including atherosclerosis, and has attracted much interest, although clinical trials to examine the preventive effect of this approach on some autoimmune diseases were unsuccessful [96]. One of the critical mechanisms for mucosal tolerance induction is supposed to be induction of several types of self-antigen-specific Tregs described above [96]. Oral tolerance induction to oxidized LDL [98] or heat shock protein 60 [99] reduced the development of atherosclerosis in *Ldlr*−/<sup>−</sup> mice, in association with increased proportion of CD4+CD25+Foxp3+ Tregs in several lymphoid organs and promoted the production of TGF-β or IL-10 in mesenteric lymph nodes upon each antigen stimulation, respectively. However, antigen-specificity of expanded Tregs was not clearly determined in these studies.

Gut microbiota are highly associated with the intestinal immunity and systemic metabolic disorders. Recent experimental and clinical studies have provided evidence for an association between gut microbiota composition and development of CVD [100]. By performing 16S ribosomal RNA gene sequencing in fecal samples from CAD patients and mechanistic studies with *Apoe*−/<sup>−</sup> mice, we have recently demonstrated that the relative abundance of *Bacteroides vulgatus* and *Bacteroides dorei* was lower in patients with CAD compared with controls, and that this microbiota protects against atherosclerosis by ameliorating endotoxemia and systemic inflammation [101]. CD4+Foxp3+ Tregs were expanded in the colon lamina propria by oral administration of mouse [102] and human [103] *Clostridium* species, the spore-forming component of indigenous intestinal microbiota, and this Treg induction resulted in attenuation of experimental colitis. As the mechanisms for induction of colonic Tregs, butyrate, one of commensal bacteria-derived short-chain fatty acids, was shown to regulate the differentiation of Tregs and ameliorate the development of experimental colitis [104]. Although no prior reports investigated the role of *Clostridium* species or microbial-derived butyrate in atherosclerosis, a recent experimental study using hypertensive mice with or without atherosclerosis has reported that treatment with another short-chain fatty acid propionate prevents cardiac damage and atherosclerosis by regulating inflammatory responses, which was mainly dependent on Tregs [105]. Collectively, recent experimental and clinical data suggest that some specific gut microbiota and microbial-derived metabolites may modulate atherosclerosis. Further investigation will contribute to the development of novel therapies to prevent atherosclerotic disease through modulation of intestinal immune system.

#### *4.4. Treatment with Antibodies and Cytokines*

Intravenously administered anti-CD3 monoclonal antibodies, effective in treating autoimmune diabetes in mice [106] and humans [107], also improve atherosclerosis in mice [108] by regulating Teff immune responses and expanding CD4+CD25+ Tregs. Oral or nasal anti-CD3 antibody administration can induce CD4+LAP (latency-associated peptide)+ Tregs

that suppress experimental autoimmune diseases in a TGF-β-dependent manner [109,110]. We orally treated *Apoe*−/<sup>−</sup> mice with anti-CD3 antibodies and observed significantly reduced plaque formation and inflammation, associated with the expansion of CD4+LAP+ Tregs and CD4+Foxp3+ Tregs in mesenteric lymph nodes [111]. As a mechanism of this suppression, we speculate that expanded Tregs may move to other lymphoid organs or atherosclerotic lesions and suppress proatherogenic immune responses. We propose the novel idea that oral immune modulation could serve as an attractive therapeutic approach to atherosclerotic disease.

CD4+Foxp3+ Tregs highly express CD25, a component of the high-affinity IL-2 receptor, and vigorously proliferate in the presence of IL-2. Recombinant mouse IL-2/anti-IL-2 monoclonal antibody complexes (IL-2 complexes) efficiently and selectively expand CD4+CD25+Foxp3+ Tregs and attenuate atherosclerosis in *Ldlr*−/<sup>−</sup> [112] or *Apoe*−/<sup>−</sup> mice [113]. Induction of CD4+Foxp3+ Tregs by IL-2 complex treatment also provides protection against angiotensin II-induced AAA formation in *Apoe*−/<sup>−</sup> mice [71]. These reports suggest that IL-2 complex treatment could be a possible strategy to prevent both atherosclerosis and AAA. Considering that suppressive functions of Tregs can be impaired under inflammatory conditions [114], we speculate that both Treg expansion and inhibition of Teff responses could be necessary to potently suppress atherosclerosis. In line with this idea, we found that the combination treatment with anti-CD3 antibodies and IL-2 complexes was effective in expanding Treg with activated phenotype and preventing atherosclerosis development in *Apoe*−/<sup>−</sup> mice [115].

Interestingly, a recent observational cohort study in patients with chronic graft-versushost disease demonstrated that low-dose IL-2 was safely administered in these patients, which was associated with marked and sustained expansion of Tregs and improvement of the disease manifestations in a substantial proportion of treated patients [116]. Another clinical study in patients with vasculitis induced by the hepatitis C virus showed that the administration of low-dose IL-2 increased the percentage of Tregs without adverse effects in all subjects and led to the improvement of vasculitis in most patients [117]. Based on the data obtained from experimental and clinical studies described above, a randomized, double-blind, placebo-controlled, phase I/II clinical trial in patients with stable ischemic heart disease and ACS (LILACS), has begun to assess the safety and efficacy of low-dose IL-2 in patients with CAD [118,119]. It is expected that interesting results of this trial will be reported in the future.

#### *4.5. Ultraviolet B (UVB)-Based Phototherapy*

It is historically clear that sunlight exposure is indispensable for the maintenance of our health. UV is categorized into UVA (320–400 nm), UVB (280–320 nm), and UVC (100–280 nm). As UVC is blocked by the atmosphere and the ozone layer, we receive UVA and UVB wavelengths from natural sunlight in daily life. UVB irradiation is known to have beneficial effects on the immune system due to its anti-inflammatory and immunosuppressive actions, although chronic excessive UVB exposure could cause skin cancer or infectious diseases [120]. Based on this, UVB-based phototherapy is an established treatment for various human skin diseases without severe adverse effects [121].

Our recent work revealed that broad-band UVB (a continuous spectrum from 280 to 320 nm with a peak around 313 nm) exposure attenuates the development and progression of atherosclerosis in *Apoe*−/<sup>−</sup> mice, associated with a systemic increase in CD4+Foxp3+ Tregs with higher suppressive capacity and a decrease in proinflammatory IFN-γ production from T cells [122]. Langerhans cells (LCs), epidermal Langerin+ DCs having an important role in presenting antigens to T cells, are reported to regulate immunoinflammatory reactions following their activation by various environmental stimuli, such as UVB [123,124]. Using LC-depleted mice on an *Apoe*−/<sup>−</sup> background, we clearly showed that LCs play an indispensable role in the systemic expansion of Tregs and attenuation of atherosclerosis development and progression, suggesting the skin immune system as a novel therapeutic target for atherosclerosis. We also investigated the effect of UVB irradiation on the development of atherosclerosis-related vascular disease, such as AAA, and found that UVB irradiation attenuated the development of angiotensin II-induced AAA under hypercholesterolemia and prevented death due to its rupture [125]. Further analysis using hypercholesterolemic DEREG mice revealed that genetic depletion of CD4+Foxp3+ Tregs abrogated the protective effect of UVB treatment, indicating an indispensable role of Tregs for UVB-mediated prevention of AAA formation.

Clinical studies in patients with skin autoimmune disease demonstrated that bathpsoralen UVA or narrow-band UVB (a narrow peak around 311 nm) therapy expanded CD4+CD25+Foxp3+ Tregs in the periphery and improved disease state [126,127]. The efficacy of UVB therapy may vary depending on its wavelengths, although the detailed mechanisms of UVB-mediated protective effects on skin diseases remain obscure. Within the UVB spectrum, narrow-band UVB is the most effective wavelength for treating psoriasis [121]. Likewise, there might be some specific UVB wavelengths that are effective in preventing atherosclerosis, although we have no information about this so far. If the issues regarding the efficacy and safety of UVB therapy are overcome, UVB phototherapy could be an attractive immunomodulatory strategy for preventing atherosclerotic CVD.

#### *4.6. Approaches to Regress Atherosclerosis*

A great number of human and animal studies have revealed mechanisms of atherosclerotic plaque development, whereas mechanisms that reverse the disease remain obscure. In a clinical study, very intensive statin therapy lowered LDL cholesterol levels and increased high-density lipoprotein cholesterol levels, resulting in atherosclerosis regression, indicating the importance of lipid control for regressing established plaques [128]. In addition, experimental studies using several mouse models of atherosclerosis regression have highlighted the involvement of immunoinflammatory reactions in the process of atherosclerosis regression [129–131], although the precise mechanisms underlying this process have not been completely elucidated. Using a new mouse model of atherosclerosis regression in *Ldlr*−/−, we found that Eicosapentaenoic acid induced atherosclerosis regression by modulating DC functions and reducing CD4<sup>+</sup> T cell numbers without increasing CD4+CD25+Foxp3+ Tregs [132], supporting the clinical benefits of this drug for the treatment of CVD in Japanese hypercholesterolemic patients [133]. Using the same mouse model, we also reported that intravenous injection of anti-CD3 antibodies induced rapid regression of established atherosclerotic lesions, associated with increased proportion of CD4+CD25+Foxp3+ Tregs in the regressing plaques as well as in the lymphoid organs [134]. This study for the first time suggested the possibility that the expansion of Tregs might contribute to regressing atherosclerotic plaques in mice. In line with this, a recent experimental study using various mouse models of atherosclerosis regression has demonstrated that CD4+CD25+Foxp3+ Tregs accumulated in the regressing plaques, and played an indispensable role for inflammation resolution in the artery wall by regulating macrophage- and T cell-mediated pro-inflammatory responses [135]. Collectively, these reports imply that in addition to intensive lipid-lowering therapies, the promotion of Treg immune responses may represent an effective therapeutic approach for atherosclerosis regression.


**Table 2.** Strategies to prevent or treat atherosclerosis by promoting regulatory immune responses.

ApoB, apolipoprotein B; DC, dendritic cell; Foxp3, forkhead box P3; IFN, interferon; IL, interleukin; LAP, latencyassociated peptide; LDL, low-density lipoprotein; Teff, effector T cell; TGF, transforming growth factor; Treg, regulatory T cell; UVB, ultraviolet B.

#### **5. Conclusions**

It has now become evident that arterial inflammation caused by immune dysregulation critically contributes to the development of atherosclerotic CVD. Recent clinical studies have confirmed that an anti-inflammatory therapy could be a possible strategy to prevent atherosclerotic CVD in patients with past cardiovascular disease history [4–6]. Although anti-inflammatory pharmacological therapies seem to be safe and effective in treating

experimental atherosclerosis in mice, general immunosuppression caused by such therapies would cause adverse immune responses in humans [3]. Moreover, it will take much cost to prescribe expensive drugs for a long time. Therefore, other strategies to dampen inflammatory responses in atherosclerosis should be considered.

Notably, firm evidence indicating the involvement of the adaptive immunity including Teff and Treg immune responses in atherosclerosis has accumulated [7]. Pharmacological approaches, vaccination strategies, and cell transfer of Tregs have been shown to reduce atherosclerosis development in mice [9,10]. Strategies to prevent atherosclerosis through boosting regulatory immune responses seem to be attractive and could be clinically applied for the treatment of atherosclerotic disease. However, despite accumulated knowledge about the role of protective Tregs in experimental atherosclerosis, we still lack direct clinical evidence to support this idea. To translate a large number of important findings obtained by animal experiments to clinical settings, extensive clinical studies will be required.

**Author Contributions:** T.T. and N.S. wrote the manuscript and contributed equally to this work. Y.R. critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by JSPS KAKENHI Grant Numbers 23790849, 25860601, 15K09156, and 18K08088.

**Acknowledgments:** We would like to thank our laboratory members for helpful discussions.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

