Red light-green light: T-cell trafficking in cardiac and vascular inflammation

Abstract

Extravasation of T cells from the bloodstream into inflamed tissues requires interactions between T cells and vascular endothelial cells, a necessary step that allows T cells to exert their effector function during the immune response to pathogens and to sterile insults. This cellular cross talk involves adhesion molecules on both the vascular endothelium and the T cells themselves that function as receptor-ligand pairs to slow down circulating T cells. These will eventually extravasate into sites of inflammation when they receive the correct chemokine signals. Accumulation of T cells within the vascular wall can lead to vessel thickening and vascular disease, whereas T-cell extravasation into the myocardium often leads to cardiac chronic inflammation and adverse cardiac remodeling, hallmarks of heart failure. On the flip side, T-cell trafficking is required for pathogen clearance and to promote tissue repair after injury resulting from cardiac ischemia. Thus, a better understanding of the central players mediating these interactions may help develop novel therapeutics to modulate vascular and cardiac inflammation. Here, we review the most recent literature on pathways that regulate T-cell transendothelial migration, the last step leading to T-cell infiltration into tissues and organs in the context of vascular and cardiac inflammation. We discuss new potential avenues to therapeutically modulate these pathways to enhance or prevent immune cell infiltration in cardiovascular disease.

INTRODUCTION

Trafficking of leukocytes from the bloodstream into sites of injury and inflammation is a crucial step for an optimal immune response, which culminates in leukocyte transendothelial migration (TEM). Innate immune cells such as neutrophils and circulating monocytes are the first responders in the acute immune response. They are the first leukocytes recruited to sites of inflammation and they release soluble factors that contribute to the initiation of the T-cell immune response. Unlike innate immune cells, T cells rely on recognition of antigens that dictate their activation, clonal expansion, and expression of adhesion molecules necessary to traffic to sites of inflammation. Neutrophils and monocytes usually precede T-cell extravasation during the acute inflammatory response. When sustained over time, constant influx of innate cells and T cells results in chronic inflammation, tissue, and organ damage. Leukocyte TEM typically occurs in postcapillary venules. In conditions such as atherosclerosis, pulmonary artery hypertension, and abdominal aortic aneurysm (AAA), it occurs in larger vessels and contributes to exacerbation of disease. T-cell accumulation in the vasculature and extravasation into nonregenerative organs such as the heart often result in fibrotic remodeling that contributes to vessel and organ stiffening, hallmarks of cardiovascular disease (1, 2). Fibrosis can be advantageous to the heart when cardiac repair is needed to prevent rupture post-ischemia, but in the long term, excessive fibrosis contributes to decreased contractility and heart failure (HF) (3, 4).

The leukocyte extravasation cascade has been previously therapeutically targeted, with limited success. A few examples include inhibition of α4 integrin, an adhesion molecule involved in leukocyte rolling and arrest, which does not protect against ischemic stroke (5); and antagonism of CCR2 with small molecules or blocking antibodies to target CCR2⁺ proinflammatory monocytes, which successfully prevents inflammation and improves systolic function in preclinical models but thus far has not yielded to FDA approved drugs (6). This highlights the importance of gaining further mechanistic insight in leukocyte trafficking mechanisms. Here, we review recent discoveries of molecular players that participate in T-cell and leukocyte TEM in hypertension and AAA associated vascular inflammation, and in cardiac inflammation in the onset of myocardial repair and adverse remodeling in HF (Fig. 1 and Table 1).

Figure 1.

A: classic players of TEM involve T-cell selectin ligands and integrins, which interact with EC selectins and ICAM-1 and VCAM-1, respectively. VE-Cad is at the cell junctions and modulates leukocyte passage across ECs (3). Endothelial cell STING controls Type I-IFN production, which signals through IFNAR and induces CXCL10, attracting CXCR3⁺ T cells (7). TRPC6 colocalizes with PECAM-1 on the surface of ECs (8) and enables an influx of cytosolic calcium that activates Calmodulin (CaM) and CAMKIIδ for leukocyte TEM (9). Mechanosensing activates EC-PIEZO-1 for subsequent leukocyte TEM (10), and macrophage and T-cell miR-34a-5p represses CXCL10 and CXCL11 mRNA and decreases CXCR3, potentially impairing TEM (11). B: in hypertension, T-cell-derived miR-214 induces proinflammatory gene expression and endothelial dysfunction (12). Activated by IgE, Nhe1 activity controls macrophage accumulation and EC adhesion molecule expression (13). Human CD16⁺CX3CR1⁺ monocytes interact with and induce the production of EC-derived CX3CL1 (14). C: activated CXCR3⁺ T cells recognize CXCL9/10 to traffic to the heart (15). Failing human hearts have decreased cardiomyocyte T-cad expression (16), increased cardiomyocyte CAMKIIδ and NLRP3 inflammasome activation that results in leukocyte myocardial infiltration (17). IL-3⁺CD4⁺ T cells stimulate IL-3R⁺ macrophages to produce monocyte attracting chemokines, and recruited monocytes differentiate into APCs that stimulate IL-3⁺CD4⁺ T-cell proliferation (18). D: global CCL17 deletion increases Treg recruitment post-MI (19), and cardiomyocyte GRK5 increases monocyte recruitment post-MI, with no effect on T-cell recruitment (20). Image created with BioRender.com and published with permission. ECs, endothelial cells; TEM, transendothelial migration; Treg, T regulatory cell.

Table 1.

Leukocyte trafficking pathways in vascular and cardiac inflammation

Emerging Molecular Trafficking Pathway/Cell Type	Inflammatory Condition/Vascular Bed	Pathway Promoting Disease, Promoting Repair, or Preventing Disease
STING	Peritoneal inflammation	Promotes disease. Endothelial STING deficiency results in decreased T-cell TEM and peritoneal inflammation (7)
TRPC6	Cremaster inflammation	Promotes disease. TRPC6 deficiency results in impaired neutrophil and monocyte TEM and prevents cremaster muscle inflammation (8)
CAMKIIδ	Dermal and cremaster inflammation Pressure overload induced heart failure	Promotes disease. Endothelial CAMKIIδ facilitates neutrophil and monocyte TEM and its deficiency results in decreased dermal and cremaster inflammation (9) Promotes disease. Cardiomyocyte CAMKΙΙδ deficiency results in decreased cardiac inflammation and fibrosis (17)
PIEZO-1	Peritoneal inflammation	Promotes disease. Endothelial cell PIEZO-1 deficiency results in impaired neutrophil and monocyte TEM and decreased peritoneal inflammation (10)
T-cell miR-214	Hypertension	Promotes disease. miR-214−/− deficiency reduces profibrotic cytokines and chemokine receptors that contribute to vessel dysfunction and stiffness (11)
Syncytin-1	Preeclampsia	Prevents disease. Overexpression reduces T-cell proliferation, which may result in decreased T-cell placental inflammation in preeclampsia (21)
Silibinin	Preeclampsia	Prevents disease. Induces the anti-inflammatory cytokine IL-10 in PBMCs from women with preeclampsia, which may dampen T-cell placental inflammation (22)
Na+H+ exchanger-1 (Nhe1)	Abdominal aortic aneurysm (AAA)	Promotes disease. Nhe1 insufficiency reduces macrophage and T-cell adhesion and recruitment to the vessel wall (13)
CXCL9/10-CXCR3	Pressure overload induced heart failure	Promotes disease. CXCR3 deficiency results in decreased T-cell adhesion to ICAM-1 and decreased cardiac inflammation and fibrosis (15)
T-cadherin (T-cad)	Nonischemic cardiomyopathy	Disease recovery. Low cardiac T-cadherin protein levels in patients correlates with high CD3 T-cell cardiac infiltration (16)
GRK5	Myocardial infarction (MI)	Prevents disease. Cardiomyocyte GRK5 deficiency decreases monocyte recruitment post-MI, with no effect on T-cell infiltration (20)
CCL17	Myocardial infarction (MI)	Promotes disease. CCL17 deficiency improves Treg-mediated healing and repair post-MI (19)
Integrin-associated protein CD47	Dermal and peritoneal inflammation Myocardial infarction (MI)	Promotes disease. CD47 deficiency impairs T-cell recruitment to dermal and peritoneal inflammatory sites (23, 24) Impairs cardiac repair. CD47 inhibition enhances macrophage phagocytosis of dead cardiomyocytes and contributes to increased infarct size (25)
CD16+CX3CR1+ monocytes	Atherosclerosis	Promote disease. CD16+CXCR1+ monocytes induce endothelial expression of adhesion molecules, proinflammatory chemokines (14)
IL3+CD4+ T cells	Myocarditis	Promote disease. CD4+ T-cell-specific IL-3 depletion prevents cardiac inflammation by decreasing monocyte recruitment to the heart (18)
CCR2+ monocytes, CCR2+ Cardiac resident macrophages	Pressure overload induced heart failure Myocardial ischemia reperfusion injury (IRI)	Promote disease. CCR2+ monocyte depletion in the onset of TAC prevents initial cardiac remodeling. Once HF is established, CCR2 depletion had no effect on cardiac remodeling (26) Promote adverse remodeling after IRI. CCR2+ depletion prior to IRI results in decreased infarct area and improved systolic function (27)
CD4+ T cells	Myocardial infarction Pressure overload induced HF and hypertension	Promote repair post-MI. CD4-deficient mice have worse outcome and increased cardiac rupture (28) Promote disease. T-cell-deficient mice have decreased adverse vascular and cardiac remodeling (1, 29)

HF, heart failure; TEM, transendothelial migration; Treg, T regulatory cell.

CLASSIC AND EMERGING NOVEL PLAYERS IN LEUKOCYTE TEM

Endothelial cells have antiadhesive properties that allow them to be resistant to leukocyte adhesion and maintain vascular homeostasis acting as active gatekeepers of leukocyte TEM. They rapidly respond to inflammatory stimuli released in tissues on sterile or pathogenic insults and transform into an adhesive surface to leukocytes through the expression of inducible adhesion molecules. These function as receptors of their counterpart adhesion molecule ligands expressed in circulating leukocytes. The description of these sequential adhesion molecule interactions has resulted in a proposed multistep model that culminates in leukocyte TEM. Endothelial selectins, E- and P-selectin, adhere to leukocyte-expressed selectin ligands that result in rolling and tethering of leukocytes in the vascular wall. This step slows down circulating leukocytes and facilitates adhesion of leukocyte integrins to the endothelial cell integrin ligands intercellular and vascular adhesion molecules 1 (ICAM-1 and VCAM-1, respectively). During this intimate contact, ICAM-1, VCAM-1, platelet endothelial cell adhesion molecule (PECAM-1) and CD99, among others, reorganize to promote TEM. The junctional molecule Vascular Endothelial cadherin (VE-Cad), as well as the cytoskeletal proteins cortactin, actin, and iqGAP reorganize in both leukocytes and endothelial cells and trigger signaling pathways culminating in TEM into sites of inflammation (Fig. 1A) (3). All leukocytes follow these sequential steps in general, but differences in responsiveness to certain chemokines and in the expression of adhesion molecules and chemokine receptors have been described to be distinct in innate cells and T cells, within T-cell types, and to be dependent on the inflammatory context. A wealth of articles have extensively and elegantly reviewed the literature in the role these classic players play in TEM and organ physiology, thus these will not be the focus of this review (3, 4). Recently, new and somehow unexpected players have been reported to contribute to TEM in ways that are dependent and independent of the classic contributors to TEM, stretching the boundaries of molecular networks that contribute to TEM. These are the focus of this section.

One of such novel players is the Stimulator of Interferon Genes (STING), a central regulator of antiviral immune responses through the production of Type-I Interferons (Type I-IFN; Fig. 1A). Global STING-deficient mice (STING^−/−) had decreased T-cell infiltration in an experimental model of TNFα induced peritonitis compared with wild-type (WT) mice (Table 1). Endothelial-cell-specific deletion of STING in mice (EC-STING^−/−) also resulted in decreased T-cell trafficking to the peritoneal cavity in response to TNF-α. In contrast, neutrophil infiltration was not impaired in EC-STING^−/− mice compared with WT mice. In vitro TEM studies under flow conditions using mouse heart endothelial cells and T cells isolated from wild-type and STING^−/− mice further demonstrated that T-cell TEM was impaired when STING was lacking in the endothelial cells but not when it was lacking on the T cells. Mechanistically, endothelial STING contributed to the release of chemokine CXCL10, a Type I-IFN responsive gene, but not to the expression of the classic adhesion molecules E-selectin, VCAM-1, and ICAM-1 (7). Although the specific role of endothelial STING in TEM has not been investigated in cardiac or vascular inflammation per se, global deletion of cGAS, a DNA sensor, which is upstream STING, results in decreased cardiac inflammation post-myocardial infarction (MI). This observation suggests the possibility that endothelial STING may contribute to leukocyte recruitment to the heart, opening a new avenue to study the physiological role of endothelial STING in T-cell TEM in cardiovascular disease (30, 31).

Another recently described contributor to leukocyte TEM is the calcium channel transient receptor potential canonical 6 (TRPC6; Fig. 1A). Although it was known for decades that increased cytosolic calcium in endothelial cells was a necessary prerequisite to leukocyte TEM, its mechanisms of action were unknown until the initial observation that TRPC6 colocalized with PECAM-1 on the surface of endothelial cells (8). In vitro knockdown of TRPC6 in endothelial cells resulted in reduction of neutrophil TEM by 40%, whereas selective activation of TRPC6 rescued this phenotype. Studies in vivo using bone marrow chimeras and real-time visualization of TEM in the cremaster muscle demonstrated that deficiency of TRPC6 in the nonmyeloid compartment impaired leukocyte TEM. Using similar in vitro and in vivo loss of function approaches, additional endothelial proteins dependent on calcium for activation were identified to contribute to TEM. These include calmodulin, and calcium/calmodulin kinase IIδ (CaMKIIδ). Calmodulin binds to the scaffold protein iqGAP in the cytosol and stabilizes cellular junctions. In response to calcium influx, calmodulin associates with CaMKIIδ, and this molecular interplay was recently described to coordinate leukocyte TEM. Indeed, inhibitory peptides and expression of dominant negative CaMKIIδ in endothelial cells significantly reduced neutrophil TEM in vitro. Moreover, mice with specific endothelial deletion of CaMKIIδ using an inducible Cre-lox system had reduced neutrophil infiltration and inflammation in the skin in experimental dermatitis (9). Aside from endothelial cells, CaMKIIδ in cardiomyocytes was shown to control cardiac rhythm and contractility (32–34), and to be activated in response to cardiac pressure overload. Moreover, cardiomyocyte-specific CaMKIIδ-deficient mice are protected from cardiac inflammation and fibrosis induced by cardiac pressure overload (17). Taken together, these reports suggest coordinated actions of cardiomyocyte and endothelial cell CaMKIIδ may contribute to leukocyte recruitment to the heart: in cardiomyocytes CaMKIIδ modulates the release of soluble factors that can activate endothelial cells (Fig. 1C), whereas in endothelial cells, CaMKIIδ participates in TEM through modulation of iqGAP (Fig. 1A; Table 1).

Endothelial cells are constantly experiencing mechanical forces imprinted by the blood flow. A recent report demonstrates that the mechanosensitive cation channel PIEZO-1 is activated by mechanical forces in the circulation and contributes to TEM (Fig. 1A). Mechanistically, activated PIEZO-1 mediates clustering of the classical TEM regulator ICAM-1 and increases cytosolic calcium influx that results in the dissociation of endothelial junctions and allows leukocyte passage. In vivo, endothelial-cell-specific deletion of PIEZO-1 using a Cre-lox system resulted in decreased peritoneal inflammation and leukocyte recruitment (Fig. 1A; Table 1) (10).

Lastly, microRNAs are also emerging as novel indirect contributors to leukocyte TEM. One example is miR-34a-5p, which was recently reported in “in silico” assays to bind to the 3′ Untranscribed regions (3′ UTRs) of chemokines CXCL10 and CXCL11 mRNA, rendering fewer properly translated proteins with functional binding sites, and less interaction with their cognate receptor CXCR3 (Fig. 1A). T cells overexpressing miR-34a-5p showed decreased surface CXCR3 expression and CXCL10/11 production. The CXCR3-CXCL10/11 axis is critical for T-cell integrin activation required for adhesion to ICAM-1 and TEM. This study may explain the lack of success of clinical trials using liposome-encapsulated miR-34a-5p to treat cancer. Although this approach may be beneficial to target the tumor itself, its effects on suppressing T-cell CXCR3 expression would result in reduced T-cell tumor infiltration and antitumor activity (11).

Taken together, new proteins with established functions in antiviral response, calcium homeostasis and mechanosensing, as well as microRNAs that can alter other proteins are emerging as novel contributors to the complex process of TEM. Although some of these were studied in the context of innate cell TEM, others seem to be specific for T cells. The cellular interplay during chronic inflammation highlights the need to investigate these pathways to understand the pathophysiology of cardiovascular disease.

T-CELL TRAFFICKING IN VASCULAR INFLAMMATION

T-cell trafficking to the vascular wall has been perhaps most extensively studied in the context of atherosclerosis, a condition characterized by cholesterol deposits in the vessel wall that lead to innate cells and lymphocyte recruitment, which, in turn, fuel plaque formation and adverse vascular remodeling. The many mechanisms involved in this process have been reviewed elsewhere and will not be the focus of this review. However, novel players of T-cell trafficking in vascular inflammation continue to emerge, not only in the context of atherosclerosis but also in the context of other cardiovascular conditions such as hypertension and AAA, also characterized by vascular inflammation in the small and large vessels.

Hypertension is characterized by vascular stiffness, elevated levels of angiotensin II (Ang II) and high blood pressure. If left untreated, it is a major risk factor for HF. A common experimental model of hypertension is to infuse Ang II in mice as a way to increase blood pressure that results in vascular stiffness and vasoconstriction. Rag^−/− mice, deficient in lymphocytes, do not develop hypertension and adverse vascular remodeling in response to Ang II. Reconstitution of Rag^−/− mice with T cells in the onset of Ang II infusion reverses this phenotype to hypertension and vascular inflammation, highlighting a central contribution of T cells in blood pressure modulation and vascular inflammation in hypertension (29, 35, 36). One recently described novel mechanism on how T cells contribute to hypertension is through T-cell-derived miR-214 (Fig. 1B; Table 1). Adoptive transfer of miR-214^−/− T cells into Rag^−/− mice in the onset of Ang II infusion resulted in reduced perivascular fibrosis as compared with adoptively transferred wild-type T cells. Mechanistically, T-cell miR-214 regulated the production of the cytokines IL-7, TNF-α, IFN-γ, and IL-9 and the expression of the chemokine receptors CCR1, CCR2, CCR4, CCR5, CCR6, CXCR3. The downregulation of these chemokine receptors in miR-214-deficient T cells suggest that T-cell miR-214 facilitates T-cell trafficking to the vessel wall as a pathological mechanism induced by Ang II in hypertension. This novel axis was validated in hypertensive patients, whose miR-214 levels were elevated and correlated with increased arterial stiffness and vessel dysfunction (12). It is possible that this is due to interactions of T cells with resident cardiac fibroblasts or endothelial cells that induce changes in cellular states prone to produce collagen and ultimately fibrosis (2).

T cells are also found in the decidua, the maternal part of the placenta, in women with preeclampsia who develop hypertension during pregnancy (37). The protein Syncytin-1, a central regulator of placental development and function, has recently emerged as a potential regulator of T-cell function in the placenta. Syncytin-1 overexpression in human placental-derived cells resulted in impaired T-cell proliferation in coculture studies (Table 1) (21). Whether Syncytin-1 may function in T-cell trafficking as it relates to placental infiltration has yet to be discovered, but this study suggests that placental Syncytin-1 may regulate the number of T cells that possess the potential to cause preeclampsia. The role of T cells in preeclampsia is further supported by studies in which peripheral blood mononuclear cells (PBMCs) from normotensive and preeclamptic pregnant women were cultured with the flavonoid Silibinin. Silibinin treatment had an immunomodulatory effect on PBMCs from preeclamptic women involving a robust secretion of the anti-inflammatory cytokine IL-10 (Table 1) (22). Although further investigation is warranted to investigate how Silibinin modulates IL-10 secretion and whether this results in decreased T cells trafficking to the placenta in vivo, these findings would suggest a new immunomodulatory way to dampen T-cell effector function in preeclampsia, and the flavonoid Silibinin could be potentially used to decrease T cells’ proinflammatory activity and protect mothers from the development of preeclampsia. Further support for a role for T-cell recruitment in hypertension in humans comes from a recent study in adolescents demonstrating elevated numbers of circulating effector and memory T cells. Specifically, a subset of T cells expressing PECAM-1 was decreased, correlating with enhanced T-cell effector function (38) and hypertensive organ damage, analyzed by pulse wave velocity and left ventricular mass index (39). Although the presence of vascular or cardiac T cells could not be evaluated in the vasculature in these studies, these data suggest that effector and memory T cells may traffic to the inflamed vasculature in adolescents and contribute to hypertension persistence and progression to organ damage.

A novel regulator of T-cell trafficking in vascular inflammation is the Na⁺ H⁺ Exchanger-1 (Nhe1), a plasma membrane-resident sodium-hydrogen transport protein that is activated by immunoglobulin E (IgE; Fig. 1B). Using a model of Ang-II infusion in hyperlipidemic mice to model AAA in mice and a pH-sensitive imaging technique, Liu et al. (13) found acidic extracellular regions in locations of high macrophage accumulation, high IgE expression, and high levels of apoptosis in human and mouse AAA lesions. Nhe1 insufficiency in mice resulted in decreased AAA incidence and size, as well as decreased recruitment of macrophages and T cells to the vascular lesion. IgE induction of endothelial cell adhesion molecule expression was also decreased, confirming that Nhe1 functions as a positive regulator of T-cell and monocyte adhesion to the vasculature that exacerbates AAA (Table 1). The cellular-specific function of Nhe1 was not depicted in this study, yet it is possible that activation of this channel by IgE results in extracellular proton deposition that activates immune cells and leads to endothelial cell adhesion molecule expression in AAA.

Vascular inflammation is also aggravated in patients with chronic kidney disease (40). Patients with atherosclerosis and kidney failure have increased CD16⁺ CX3CR1⁺ patrolling monocytes and their ligand CX3CL1 is increased in the atherosclerotic plaques. Mechanistic in vitro coculture studies demonstrate that CD16⁺ CX3CR1⁺ monocytes from these patients induce downregulation of genes involved in vasodilation in endothelial cells, supporting more vascular stiffness, and the relevance of vascular inflammation beyond atherosclerosis (Fig. 1B; Table 1) (14).

Taken together, T-cell accumulation in the vascular wall of small and large vessels is associated with adverse vascular remodeling. Investigating novel regulators of T-cell trafficking to the vascular wall and complementing mechanistic studies with vascular function read outs in patients will bring new light to our understanding of the role of inflammation in vascular pathophysiology.

T-CELL TRAFFICKING IN CHRONIC CARDIAC INFLAMMATION

Hypertension, if untreated, often leads to nonischemic HF, a deadly syndrome characterized by systemic and cardiac inflammation. Some of the well-established preclinical models of HF are indeed induced by Ang-II infusion and by surgically inducing cardiac pressure overload by transverse aortic constriction (TAC). These models, combined with the use of human cardiac tissue from patients with HF, have led to the discovery that T cells traffic to the heart in HF (1). Patients with HF have increased circulating levels of chemokines CXCL9 and CXCL10 as well as the soluble adhesion molecule ICAM-1 (15, 41). The specific functional role of these chemokines and adhesion molecules in HF has been investigated in mice: ICAM-1 expression is significantly increased in the hearts of mice subjected to TAC and to Ang II induced hypertension (42, 43). TAC induces the production of CXCL9 and CXCL10 in cardiac macrophages and fibroblasts, which in turn attract CXCR3⁺ T cells to the heart. Studies in CXCR3-deficient mice further corroborated a role for this receptor in T-cell infiltration in the heart. Moreover, in vitro studies demonstrated that CXCR3 engagement of CXCL9 and CXCL10 was essential for T-cell adhesion to ICAM-1 (Fig. 1C; Table 1) (15). An important correlation observed in humans is that circulating CXCR3⁺ T cells in patients with nonischemic HF express more lymphocyte function-activated antigen -1 (LFA-1), the ligand for ICAM-1, and that T cells isolated from patients with nonischemic HF adhered in higher numbers to activated primary endothelial cells under shear stress conditions in vitro than those from healthy volunteers (1). This, together with the presence of CXCR3⁺ T cells in the hearts of patients with end-stage nonischemic HF, supports a heightened T-cell ability to traffic to the heart (15).

CCR2⁺ macrophages were found to precede T-cell cardiac infiltration in response to TAC. Antagonism of CCR2 in the onset of TAC revealed that while infiltrating, CCR2⁺ monocytes contribute to adverse remodeling (26). Using lineage tracing to track macrophages, a recent study demonstrated that CCR2⁻ and CCR2⁺ cardiac resident macrophages of yolk sac and bone marrow origin, respectively, populate the heart. These showed differential enrichment of proinflammatory genes, and also differences between CCR2⁺ resident macrophages and CCR2⁺ infiltrating monocytes after injury. Deletion of CCR2⁺ and CCR2- macrophages before cardiac ischemic reperfusion injury (IRI) using genetic strategies resulted in decreased and increased infarct size, respectively. These findings demonstrated a differential role for resident macrophages in postinfarction remodeling (Table 1) (27). With the development of single-cell RNA sequencing techniques, more immune subpopulations are being identified in healthy and diseased human and mouse hearts. Correlations between transcriptional changes in TAC-induced HF and patients with HF further support that T-cell and other immune cell trafficking to the heart directly correlates with cardiac pathology (44, 45). Protein validation of the wealth of RNA sequencing data available for low proportion immune cell populations in the heart is the immediate next step to confirm these interesting novel observations at the transcriptome level.

A recent study investigated the protein hormone adiponectin and its receptor T cadherin (T-cad). Adiponectin is a circulating serum biomarker hormone produced by adipocytes. It accumulates in heart, vascular endothelium, and skeletal muscles through its interaction with T-cad, primarily expressed on the luminal surface of endothelial cells and, to a lesser extent, in vascular smooth muscle cells (46). A recent study analyzed endomyocardial biopsies of nonischemic dilated cardiomyopathy patients over the course of 5 yr. The authors found that adiponectin and T-cad are abundantly expressed in the heart and blood vessels in healthy patients and reported an inverse correlation between T-cad with cardiac function. In the context of cardiac inflammation, low cardiac T-cad levels correlated with high CD3⁺ T-cell counts in the cardiac histological sections, suggesting a potential role for T-cad in modulating cardiac T-cell infiltration (Fig. 1C; Table 1) (16).

A frequent cause of dilated cardiomyopathy in children and young adults is myocarditis, a condition generally triggered by cardiotropic infections that subsequently enhance a cardiac autoimmune response, characterized by abundant T-cell infiltrates in the heart (6). T cells expressing IL-3 (IL-3⁺ CD4⁺ T cells) were recently described to infiltrate the heart and cause autoimmunity in mice. Initially described as a growth factor required for hematopoiesis, IL-3 has emerged as a T-cell cytokine that enhances antigen presentation to T cells in bacterial infections and autoimmunity. A recent study demonstrates the presence of IL-3⁺ CD4⁺ T cells in the heart in autoimmune myocarditis. IL-3⁺ CD4⁺ T cells from mice with myocarditis stimulated the IL-3 receptor (IL-3R) in cardiac macrophages, which, in turn, produced monocyte-attracting chemokines. Infiltrating monocytes not only caused cardiac inflammation by releasing proinflammatory cytokines but also by further stimulating IL-3⁺ CD4⁺ T-cell proliferation within the heart (Fig. 1C; Table 1). The fact that IL-3-deficient mice and anti-IL-3 therapy ameliorated disease further emphasizes the role of the IL-3-IL3R axis in leukocyte cardiac infiltration (18).

Identifying the cellular and molecular contributors to cardiac leukocyte trafficking is the next step toward developing immune modulatory therapies to protect the heart from adverse cardiac remodeling.

T-CELL TRAFFICKING TO PROMOTE CARDIAC HEALING

A major complication of atherosclerosis is plaque rupture in heart vessels leading to MI. Immune cell trafficking to the site of injury is critical for survival as it promotes cardiac repair and the scar required to prevent rupture. For example, CD4⁺ T cells are expanded in the cardiac draining lymph nodes of mice subjected to MI, and CD4⁺ T-cell-deficient mice had worse outcomes to MI and increased cardiac rupture. In support for a role of T cells in cardiac repair, mice lacking CD11c⁺ cells, predominantly antigen presenting cells to T cells, had a very similar phenotype, with more rupture and deteriorated left ventricular function (Table 1) (47). The multiphasic cardiac leukocyte recruitment sequence necessary for cardiac repair is initiated by early neutrophil and monocyte recruitment followed by CD4⁺ T cells, and it has been elegantly and extensively reviewed (28, 47), yet novel mechanisms continue to emerge.

One such novel regulator is the G protein-coupled receptor kinase 5 (GRK5), highly expressed in the heart and upregulated in the human failing myocardium. Besides its canonical kinase role of phosphorylating and inducing receptor recycling, GRK5 can also translocate to the nucleus to exert kinase-independent effects. Using cardiomyocyte GRK5 overexpression and gene knockout strategies, GRK5 was shown to contribute to leukocyte recruitment to the heart at both early and late time points post-MI (Fig. 1D). GRK5 deficiency resulted in reduced monocyte and neutrophil trafficking to the heart 4 days post-MI, but did not affect T-cell infiltration. This resulted in increased cardiac contractility and survival. GRK5’s inability to modulate T-cell recruitment may explain the absence of cardiac rupture despite the decreased leukocyte recruitment to the infarct zone. GRK5 overexpression had the opposite effect. At the chronic phase post-MI (8 wk), cardiomyocyte GRK5 overexpression led to increased T-cell infiltration in the infarct zone (Table 1) (20).

CCL17, expressed in monocytes and dendritic cells, has emerged as a novel contributor to adverse remodeling post-myocardial infarction by suppressing the recruitment of T regulatory cells (Tregs) to the infarct zone. Deletion of CCL17 had beneficial effects post-MI that resulted in increased trafficking of Tregs to the heart and improved healing and systolic function (Fig. 1D; Table 1) (19). Moreover, a separate study identified Integrin-associated protein CD47, widely expressed in cells of both hematopoietic and nonhematopoietic origin, contributes to T-cell TEM in vitro and CD47-deficient mice have impaired T-cell recruitment to sites of dermal and peritoneal inflammation (23, 24). Interestingly, global inhibition of CD47 in mice resulted in decreased infarct size. This was due to enhanced macrophage phagocytosis of dead cardiomyocytes following ischemia (Table 1) (25). Given the role of CD47 in T-cell TEM, it is possible that CD47 additionally contributes to better outcomes post-MI due to preventing late recruitment of T cells that participate in adverse cardiac remodeling.

Taken together, trafficking of immune cells is essential for cardiac repair post-ischemia. Novel regulators of leukocyte recruitment such as CD47, GRK5, and CCL17 continue to emerge and offer the possibility to develop approaches to either enhance or prevent leukocyte trafficking with chemical or small molecule inhibitors.

CONCLUDING REMARKS

With the discovery of new regulators that directly or indirectly contribute to immune cell trafficking to the vasculature and the heart, new avenues exist to target such pathways with potential therapeutic implications on vascular and cardiac pathophysiology. Some approaches are already available and require further testing to prove efficacy in vascular and cardiac inflammation, whereas other approaches for recently identified potential targets are still at its infancy in discovery. Phosphodiesterase 4 (PDE4) inhibitors have been developed for psoriasis and have immunomodulatory anti-inflammatory effects. In vitro studies treating endothelial cells with the PDE4 inhibitor Apremilast revealed reduced adhesion and TEM of monocytes across TNF-α-activated endothelial cells through mechanisms that involved inhibition of endothelial cell NFκΒ and MAPK signaling (48). Another PDE4 inhibitor, Tanimilast, was found to decrease dendritic cell release of Th1 cell polarizing cytokines IL-12, suggesting T-cell function can be altered indirectly through innate cells (49). Given the cross talk between innate and adaptive immunity in cardiovascular disease, these may be good therapeutic approaches in this setting. Previously developed small molecule GRK5 inhibitors, initially described for broad uses in cardiovascular disease (20), could potentially be specifically useful post-MI, based on recent findings demonstrating a role for cardiomyocyte GRK5 in cardiac repair (50). A GRK5 inhibitor could limit harmful proinflammatory leukocyte infiltration early to the infarct zone, while having no effect on T-cell infiltration, allowing T cells to promote cardiac healing quickly following the cardiovascular event. In addition, chemokine blockers or their chemokine receptor antagonists could potentially be explored for CCL17 in MI to enhance Treg trafficking and for CXCL10 to prevent Th1 cell trafficking in hypertensive HF. Contributing to existing knowledge of known classic modulators of T-cell trafficking whereas uncovering novel regulators is critical for understanding the mechanisms of inflammation in vascular and cardiac disease. Moreover, a combination of mechanistic studies in preclinical models with data from human tissue samples and functional cardiac and vascular physiological studies is warranted to make advances in our understanding of T-cell trafficking in cardiovascular disease.

GRANTS

This work was supported by NIH Grant R01 HL144477 (to P.A.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.S. prepared figures; E.S. and P.A. drafted manuscript; E.S. and P.A. edited and revised manuscript; E.S. and P.A. approved final version of manuscript.