Emerging Role of Angiotensin AT₂ Receptor in Anti-Inflammation: An Update

Abstract

The hyperactive RAS and inflammation are closely associated. The angiotensin-II/AT₁R axis of the RAS has been explored extensively for its role in inflammation and a plethora of pathological conditions. Understanding the role of AT₂R in inflammation is an emerging area of research. The AT₂R is expressed on a variety of immune and non-immune cells, which upon activation triggers the release of a host of cytokines and has multiple effects that coalesce to anti-inflammation and prevents maladaptive repair. The anti-inflammatory outcomes of AT₂R activation are linked to its well-established signaling pathways involving formation of nitric oxide and activation of phosphatases. Collectively, these effects promote cell survival and tissue function. The consideration of AT₂R as a therapeutic target requires further investigations.

1. INTRODUCTION

The RAS plays a pivotal role in the pathogenesis of hypertension and various renal and cardiovascular diseases. Ang-II is a major hormone of the RAS and acts via AT₁R and AT₂R. There is well-documented evidence suggesting the role of AT₁R in various diseases, including its proinflammatory role and its involvement in hypertension and tissue injury. AT₂R later surfaced, owing to the anti-inflammatory effects in immune [1, 2] and non-immune cells [3, 4]. During pathological conditions, a variety of immune cells infiltrate to the effector organs and modulate the organ microenvironment by releasing an array of cytokines, chemokines and effector molecules. These infiltrated cells (mainly granulocytes, lymphocytes and monocytes) interact with the resident cells via chemokines and cytokines and modulate the site of injury initially as a pro-inflammatory deleterious phase followed by an anti-inflammatory repair phase. The RAS has considerable influence on immune cells (adaptive as well as innate), injury and repair.

In general, AT₂R expression is lower as compared to its counterpart, AT₁R [5], but strongly upregulated following tissue injury [6]. Since the discovery of orally bioavailable selective AT₂R agonist C21, the anti-inflammatory role of AT₂R has become increasingly clear [7]. The goal of this article is to review the expression and function of AT₂R in response to pharmacological activation in various immune and non-immune cells and their relationship with organ-specific effects and BP regulation. Fig. (1) summarizes the balance between pro- and anti-inflammatory responses which are differentially regulated by Ang II.

Fig. (1).

Balance between pro- and anti-inflammatory responses regulated by Ang-II.

1.1. Adaptive Immune System

T-cells are the central players of an adaptive immune system. Various T-cells (CD4/CD8) play a pivotal role in pathogen/tumor clearance and maintenance of immune homeostasis. AT₂R expressing CD4⁺ T-cells were elevated in heart failure in human and rodent model. These AT₂R⁺CD4⁺ T-cells expressed FoxP3 (regulatory T-cells), secreted IL-10 (a major anti-inflammatory cytokine) and other anti-inflammatory cytokines [1]. In vivo AT₂R stimulation also leads to an increase in AT₂R⁺ cell population in the infarcted myocardium and reduced apoptosis of cardiomyocytes in rats with acute myocardial infarction [6]. Additionally, AT₂R expression was also detected in a population of CD8 T-cells infiltrating in the periinfarct myocardium. A subset of CD8⁺AT₂R⁺ was detected seven days after myocardial infarction in rats. These cells showed upregulated anti-inflammatory IL-10 and downregulated pro-inflammatory IL-2 and IFN-γ [2], which was thought to be a part of the protective response to counter AT₁R mediated aggravation of cardiomyocytes.

Recently, a population of CD4⁺AT₂R⁺ cells was identified in thoracic aortic aneurysm, a progressive fatal aortic pathological dilation. These cells displayed an inhibitory effect on proliferation and MMP2 expression in endothelial cells as opposed to the CD4⁺AT₂R⁻ cells which promoted proliferation and MMP2 expression in endothelial cells [8]. AT₁R through the activation of immune cells induces arterial inward remodeling and thus reduced blood flow in cardiovascular disorders. On the contrary, AT₂R expressing T lymphocytes, by secreting IL-17, have been involved in flow (shear stress) mediated outward remodeling, collateral arteries growth in ischemic diseases and revascularization. Mice lacking AT₂R or athymic mice (lacking T-cells) did not observe flow (shear stress) mediated outward remodeling [9].

The role of other immune cells like B cells, NK cells and NKT (natural killer T) cells has rarely been studied in renal or cardiac injury model. However, the AT₁R and AT₂R expression on uterine NK cells and their role in BP management during pregnancy in mice have been reported [10]. This further supports the notion that T-cells also have a role to play in renal, cardiac and neuronal pathologies resulting from stroke or ischemic attack. This may pave the ways to further explore the possibilities of studying AT₂R expression on immune cells in pathological conditions such as in chronic and acute renal injuries. Also, we have substantial evidence that AT₂R signaling can be modulated using receptor-ligand interactions as studied by our lab and many others [3, 11–13]. Another study provides evidence that T-cells and NK cells possess a fully functional RAS, and the ability to produce and deliver Ang-II to inflammatory sites. It revealed the presence of renin, its receptor, angiotensinogen and ACE in these cells by mRNA analysis. Both the AT₁R and AT₂R are expressed, and serve their functions in chemotaxis, cell proliferation and calcium signaling. However, antagonists specific to these receptors could not completely abolish the Ang-II-mediated effects, which indicates that another functional angiotensin receptor might be present in T-cells and NK cells and involved in these effects [14].

1.2. Innate Immune System

The innate immune response acts as the first line of defense against an array of pathogens. It is non-specific, and acts rapidly, beginning within a few hours of infection [15]. The major components of the innate immune system include physical barriers, such as epidermis of the skin, tears, saliva, mucus membranes and their secretions, and cells such as macrophages, DCs and neutrophils, which are the major phagocytic cells of the immune system. Besides, leukocytes including basophils and eosinophils are also important for pathogen clearance and immunoregulation [16]. Research establishing a link between innate immune system and hypertension has flourished in the recent years. While the role of AT₁R present on the immune cells has been sufficiently discussed in the past, there is only a limited discussion on the role of AT₂R in this context [17–18].

1.2.1. Monocytes/macrophages

Almost two decades ago, it was reported that macrophages express all the components of RAS, except chymase. A 6-fold increase in AT₂R expression was found during the differentiation of monocytes to macrophages which may be involved in the development of atherosclerosis [19]. AT₂R was upregulated in the glomerular cells and macrophages, thereby attenuating acute glomerular lesions in glomerulonephritis [20]. Another study on adjuvant-induced arthritis rat model showed that direct AT₂R stimulation with agonist CGP42112 in vitro inhibited the activity of IL-1β-stimulated monocytes, along with AT₁R downregulation and AT₂R upregulation in the stimulated monocytes [21]. The anti-inflammatory role of AT₂R activation by its agonist C21, in TLR-4 mediated inflammation indicated a critical role for IL-10. This study also provides evidence regarding the involvement of ERK1/2 pathway in the release of IL-10 following AT₂R stimulation in macrophages [22]. In a similar study, the direct activation of AT₂R on THP-1 and U937 cells by agonist C21 was anti-inflammatory since it attenuated the early inflammatory responses mediated by TLR-4, through the regulation of pro- and anti-inflammatory cytokines at both the gene and protein level [23]. In an experimental mice model of AE, direct activation of AT₂R by agonist C21 inhibited the activation of resting microglia, and infiltration of the activated microglia (a subpopulation of macrophages in the brain) in the lumbar spinal cord [24]. It was confirmed by this study that AT₂R was expressed in microglia, T-cells, spinal cord and brain aggregates.

In contrast to the anti-inflammatory role of AT₂R, peripheral macrophages expressing AT₂R predominantly infiltrate the site of nerve injury and are critical for triggering pain sensitization. Selective AT₂R antagonism by the depletion of peripheral macrophages chemo-genetically and transplantation of AT₂R-null hematopoietic cells was found beneficial in inhibiting the neuropathic pain hypersensitivity [25]. In another study by the same group of researchers, it was observed that the activation of AT₂R on peripheral/skin macrophages that infiltrate the injury site, triggers the production of ROS and RNS. This mediated the trans-activation of cell damage sensing ion channel TRPA1, resulting in the excitation of mouse and human sensory dorsal root ganglion which represents a peripheral mechanism for the induction of chronic neuropathic pain [26]. This ROS and RNS production was scavenged by AT₂R antagonist PD123319 and N-acetyl cysteine, which indicates a mechanism for the attenuation of nociceptor excitation to provide relief from pain.

Considerable evidence suggests that AT₂R has antihypertensive, anti-inflammatory and anti-proliferative effect, but some of the above mentioned recent studies highlighting the role of macrophage AT₂R in promoting chronic neuropathic pain hypersensitivity would impose a challenge on the existing knowledge about AT₂R, and necessitates elucidating other properties of this receptor, which may have not been addressed yet.

1.2.2. Dendritic Cells

For the first time, the differential expression of RAS components was identified in immature and mature DCs by using human cDNA microarray (27). The blockade of AT₂R resulted in the development of DCs expressing significantly higher levels of CD1a, a well-characterized marker for DCs differentiation, with high endocytic capacity and allostimulatory activities, as compared to control DCs (28). It was also reported that DCs sufficiently express the components of RAS including the two axis: ACE-Ang II-ATjR and the ACE2-Ang-(1–7)-MasR. Phosphorylation of ERK1/2 induced by Ang-II was markedly enhanced by Ang-(1–7) co-treatment, but this effect was significantly inhibited by AT₂R antagonist PD123319 [29]. In another study, the importance of Ang-II in the regulation of DCs was highlighted as being pro-inflammatory and immunomodulatory in function. The proliferation and phagocytic activity of DCs was markedly inhibited by Ang-II treatment, but the maturation of DCs and their migratory activity were significantly enhanced. However, it did not indicate any specific role of AT₂R in the maturation or proliferation of DCs [30].

1.2.3. Neutrophils

Neutrophils overexpressing ACE were more efficient in killing methicillin-resistant Staphylococcus aureus during infection, probably due to increased production of ROS, especially the superoxide radical generation by neutrophils. This indicates that neutrophils overexpressing ACE could boost the immune response to bacterial infections which are antibiotic-resistant [31]. However, the specific role of AT₂R in these studies was not reported. Since AT₂R is important for Ang-II induced mechanical hypersensitivity, Ang-II-induced infiltration of macrophages and neutrophils in the hind paw of the mouse was investigated for AT₂R expression. However, no amplification for either AT₁R or AT₂R could be seen in peritoneal PMN [26]. In another study, human neutrophil and platelet content, and release of 20-HETE, which is synthesized in response to Ang-II and essential for regulating vascular tone, BP and renal function, was significantly enhanced by Ang-II treatment predominantly via AT₂R. The receptor blockade by its antagonist PD123319 markedly reduced both the content and release of 20-HETE from neutrophils and platelets [32]. These findings related to the role of AT₂R in innate and adaptive immunity have been summarized in Fig. (2).

Fig. (2).

Role of AT₂R in innate and adaptive immunity.

1.3. Non-immune Cells

1.3.1. Vascular Endothelial Cells

Endothelial cells harbor and release a number of factors involved in (patho)physiology of hypertension. NO is one of these factors critically involved in vasodilation. Endothelial dysfunction, regardless of the type of injury leading to decreased bioavailability of NO, contributes to change in cell surface electrostatic charge and increases permeability to proteins and inflammatory cells [33]. AT₂R is strongly linked to NO formation [34, 35] which is corroborated by studies showing exacerbation of oxidative stress and injury in AT₂R/ApoE-double KO mice [36] and reduced expression of inflammatory mediators in atherosclerotic plaque during overexpression of AT₂R [37, 38]. C21 also reduces aortic infiltration of immune cells and vascular stiffness by reducing ECM components (fibronectin, collagen) [39]. Additionally, anti-inflammatory effects of AT₂R may encompass suppressed expression and signaling of lectin-like oxidized low-density lipoprotein scavenger receptor-1, a scavenger receptor of ox-LDL [38, 40] which requires further elucidation.

Obesity is characterized by chronic low-grade inflammation and is associated with impairment in sodium excretion. Accumulation of sodium is also linked to endothelial glycocalyx damage and impairment in vascular permeability [41]. We have observed infiltration of CD68⁺ monocyte/macrophage in obese kidney which was reduced considerably with AT₂R agonist C21 treatment [42]. As AT₂R function is linked to NO formation and sodium excretion [43, 44], the AT₂R-mediated reduction in immune cell infiltration may partly involve reduction in sodium accumulation and preservation of vascular permeability.

1.3.2. Vascular Smooth Muscle Cells

The Ang-II is a potent stimulator of cytokine release and expression of MCP-1 and other mediators (ICAM-1, VCAM-1, MMP, plasminogen activation inhibitor) involved in vascular inflammation through NFκB activation. The AT₂R function is associated with activation of phosphatases. The expression of AT₂R is transcriptionally upregulated during vascular injury. This suggests that pharmacological activation can be targeted to contain inflammation. Additionally, anti-inflammatory effects of AT₂R may also partly involve relaxation of vasculature that involves NO formation, inhibition of ROS and RhoA-Rho kinase-dependent myosin light chain phosphorylation and modulation of calcium sensitivity [45].

Tyrosine and serine phosphorylation of STATs is critical for its activation and IFN-γ signaling. IFN-γ and many other signaling molecules (IL-1β, Ang-II, glucose and insulin) upregulate AT₂R expression through interferon regulatory factor-1 [46]. Hence, AT₂R is speculated to have immunomodulatory function in VSMCs. This finding has been supported by another study performed in PC12W cells that expresses AT₂R but not ATiR. AT₂R stimulation by CGP42112 reduced STAT phosphorylation and TNF-α production [47].

1.3.3. Renal Epithelial Cells

RPTCs are vulnerable to a number of inflammatory stimuli including hypertension, proteinuria, endotoxemia (lipopolysaccharide), ischemia, glucose, fatty acids, etc.

1.3.3.1. Hypertension

Sodium may accumulate with or without fluid accumulation in the renal interstitium [48]. Intake of HSD [43] and experimental hypertensive simulations (e.g. 2-kidney-1-clip) have been associated with angiotensinemia. Ang-II is reported to facilitate sodium reabsorption which may further accelerate hypertensive phenotype [49]. Ang-II also may directly induce expression of cytokines (IL-1β, IL-6, IL-8, TNF-α) in epithelial cells [69]. Particularly in 2-kidney-1-clip hypertensive model, the AT₂R agonist C21 treatment reduced mRNA and protein expression of cytokines (TNF-α, IL-6 and TGF-β1) in renal interstitial fluid [50]. In stroke-prone SHR rats, C21 reduced ED1⁺ monocytes/macrophages infiltration, expression of collagen and tubular damage [51].

1.3.3.2. Proteinuria

Diet low [52] or rich [53] in sodium can modify immune response and kidney disease progression. Proteinuria is frequently associated with HSD intake and is an indicator of several comorbid conditions including obesity, diabetes and hypertension. The exposure of RPTCs to luminal protein (albumin) load per se has been reported to activate PKC-NOX-NFκB pathway [54] and aid in release of potent pro-inflammatory mediators from epithelial cells and influx of leukocytes causing injury and apoptosis [55]. AT₂R is located in RPTCs and has been reported to be upregulated during obesity [56]. Pharmacological stimulation of AT₂R by CGP42112a has been reported to reduce inflammatory mediators in obese rat kidney [57]. Likewise, another selective AT₂R agonist C21 has been reported to reduce proteinuria [51, 58] and several other inflammatory stimuli including Ang-II [59,60], hypertension [60, 61] and oxidative stress [57, 58]. In another study, anti-proteinuric and anti-oxidative benefits upon chronic treatment with C21 were correlated with anti-inflammatory changes in STZ-induced diabetic kidney [62].

1.3.3.3. Endotoxemia

In HK-2 cells, we have reported that AT₂R stimulation via NO formation stimulates the release of anti-inflammatory IL-10, which may have reduced the release of pro-inflammatory cytokines (TNF-α, IL-6) upon endotoxemic challenge with LPS [42]. This change is paralleled in obese Zucker rat (a model of low-grade endotoxemia), showing reductions in circulating and renal TNF-α and IL-6 upon C21 treatment [42]. Moreover, acute administration of C21 has been reported to increase renal IL-10, which is hypothesized to reduce renal content of MCP-1 and IL-6 and limit infiltration of CD11b⁺ leukocytes in mice kidney challenged with LPS [3].

1.3.3.4. Ischemia

The anti-inflammatory role of AT₂R in animal models of ischemic renal injury has been recognized based on early findings showing aggravation of renal interstitial infiltration and activation of macrophages and fibrocytes upon deletion of AT₂R [63]. Along the same line, pharmacological activation of AT₂R by C21 demonstrated reduced myeloperoxidase activity (marker of neutrophil influx) and circulating MCP-1 in rats subjected to myocardial ischemia [64].

Ischemia also causes cell cycle arrest in RPTCs. Such cells are prone to produce and release growth factors (i.e. TGF-β1, MCP-1) which through paracrine signaling, can activate and transform adjacent pericytes and fibroblast to myofibroblast with marked upregulation of collagen [65]. RPTCs may also acquire mesenchymal characteristics (secretory, motility and plasticity), undergo remodeling and transform into fibroblasts via multiple pathways in response to numerous stimuli [65–67]. In HK-2 cells, AT₂R is co-localized with TGF-βRII and CGP stimulation strengthens AT₂R-TGFβRII interaction, reduces TGFβRII expression and prevents epithelial-to-mesenchymal transition in NO-dependent manner [68]. Renal tubules may directly release these stimuli and participate in propagation of oxidative stress, activation of inflammasome, recruitment of immune cells and formation of ECM. The latter involves either synthesis or inhibition of breakdown. The breakdown process is dependent on a balance between MMP and tissue inhibitor of MMP [67]. However, the mechanism through which AT₂R reduces ECM is not known and needs to be explored.

1.4. Pulmonary Epithelial Cells

Anti-inflammatory effects of AT₂R were estimated to extend to lung parenchyma based on reports showing aggravated acid-induced or sepsis-induced acute lung injury [69]. This finding has been corroborated by a report showing reduced monocytic, neutrophilic and eosinophilic infiltration and lung injury in neonatal rat pups exposed to hyperoxia upon administration of novel and highly specific AT₂R ligand MOR107 [70]. Hyperoxia is a stimulus associated with inflammation and oxidative stress in respiratory diseases [71]. However, there were some inconsistencies as the anti-inflammatory effects of AT₂R agonist MOR107 did not correlate with mRNA expression of potent chemoattractants (MCP-1, chemokine-induced neutrophilic chemoattractant-1) and other indices of lung injury such as alveolarization, vascularization and capillary alveolar leakage [70].

1.5. Neuronal Cells

1.5.1. Ischemic Stroke

The potential of AT₂R as an anti-inflammatory target emerged from a study showing greater ischemic injury and neurological deficits in AT₂R-KO mice as compared to wild-type mice and ATiR blockade was less effective in containing injury in AT₂R-KO mice as compared to wild-type mice [72]. Pharmacological activation of AT₂R reduced the activation of microglia (local antigen presenting cell), apoptosis, infarct volume and motor deficits, and increased neuronal survival in a BP and CBF-independent manner in a focal reperfusion model of stroke-induced in conscious SHR by administering endothelin-1 (inflammatory vasoconstrictor) to the middle cerebral artery through a surgically implanted cannula [73–75]. However, one study showed improvement in CBF with C21 treatment after stroke induction by middle cerebral artery occlusion [76]. AT₂R-coupled vasodilatory NO can modulate neuronal bioavailability of free radicals [77]. Hence, anti-inflammatory effect of AT₂R in microglia can be ascribed to cerebral vasorelaxation [75], phosphatase-mediated direct inhibition of PKC activation and p47phox phosphorylation [78] and superoxide formation [74, 76]. The neuronal survival exerted by AT₂R in animals subjected to cerebral ischemia is partly dependent on hypoxia-inducible factor-α [79], brain-derived neurotrophic factor [24, 79], and vascular endothelial growth factor, which is involved in the regenerative processes [80]. These effects are AT₂R-specific as PD123319 totally [75, 78, 80] or partially reversed [73, 74]. In another study, peripheral administration of C21 after stroke induction reduced the inflammatory markers in cerebral cortex 24-hr post-ischemic stroke [81]. Moreover, the role of AT₂R in revascularization [82], in improving regenerative efficacy and migration of bone marrow mononuclear cells [83, 84] and mesenchymal stem cells [85, 86], and in cell survival [6, 83, 84] has been established.

The anti-inflammatory attributes of AT₂R in non-immune cells are summarized in Table 1.

Table 1.

Role of AT₂R as anti-inflammatory in non-immune cells.

Non-immune Cell Types	AT2R Anti-Inflammatory Functions		References
Vascular endotdelial cells	Reduced aortic immune cell infiltration Decreased fibronectin and collagen Reduced vascular stiffness Reduced CD68+ monocyte/macrophage in obese kidney		[39, 42]
Vascular smooth muscle cells	Decreased ROS, enhanced NO bioavailability, myosin light chain phosphorylation, modulation of Ca2+ sensitivity, Reduced STAT phosphorylation		[45, 47]
Renal epithelial cells	Hypertension	Reduced IL-6, TNF-α and ED1+ monocyte/macrophage infiltration Reduced tubular damage	[50, 51, 69]
	Endotoxemia	Increased NO formation and IL-10, reduced CD11b+ leukocytes infiltration in AKI	[3, 42]
	Ischemia	Decreased neutrophilic influx, MCP-1 and inflammasome activation	[64, 67]
Pulmonary epithelial cells	Reduced monocytic, eosinophilic and neutrophilic infiltration during lung injury		[70]
Neuronal cells	Decreased PKC activation and enhanced NO bioavailability Reduced superoxide formation Cerebral vasorelaxation during ischemic stroke		[74, 75, 78]

CONCLUSION

The anti-inflammatory function of AT₂R has been sufficiently evidenced by its involvement in modulating numerous key mediators (Ang-II, cytokines, chemokines, and oxidative stress) that establishes communicative pathways amid non-immune cells (neuronal, epithelial, endothelial and smooth cells) and cells of innate and adaptive immunity. The anti-inflammatory effects of AT₂R seem to be BP-independent [39, 50] and involve NO formation and activation of phosphatases as an underlying mechanism. The benefits of AT₂R activation are not only limited to hypertension, CKD, obesity, diabetes, pulmonary arterial hypertension and atherosclerosis [53] but extend to retinal diseases [87] and autoimmune diseases such as encephalomyelitis [24, 88, 89] and rheumatoid arthritis [90]. Anti-inflammation mediated by AT₂R activation is promising in myocardial infarction and cardiac repair [64, 84]. However, further studies are needed to investigate the therapeutic utility of AT₂R since macrophage AT₂R promotes chronic neuropathic pain hypersensitivity.

LIST OF ABBREVIATIONS

ACE

Angiotensin converting enzyme

AE

Autoimmune encephalomyelitis

AKI

Acute kidney injury

Ang-I

Angiotensin-I

Ang-II

Angiotensin-II

ARBs

Angiotensin receptor blockers

AT₁R

Angiotensin-II type 1 receptor

AT₂R

Angiotensin-II type 2 receptor

AT₂R-KO

Angiotensin-II type 2 receptor knock out

BP

Blood pressure

CBF

Cerebral blood flow

CKD

Chronic kidney diseases

DCs

Dendritic cells

ECM

Extracellular matrix

ERK

Extracellular signal-regulated kinase

20-HETE

20-hydroxyeicosatetraenoic acid

HK-2

Human kidney-2

ICAM-1

Intracellular cell adhesion molecule-1

IL-1β

Interleukin-1β

IL-10

Interleukin-10

IL-6

Interleukin-6

MCP-1

Monocyte chemoattractant protein-1

MMP

Matrix metalloproteinase

NFκB

Nuclear factor kappa B

NK

Natural killer

NKT

Natural killer T lymphocytes

NO

Nitric oxide

NOX

NADPH oxidase

ox-LDL

Oxidized LDL

PMN

Polymorphonuclear

RAS

Renin-angiotensin system

ROS

Reactive oxygen species

RPTCs

Renal proximal tubular epithelial cells

RNS

Reactive nitrogen species

SHR

Spontaneously hypertensive rat

STAT

Signal transducers and activators of transcription

TGF-β1

Transforming growth factor-β1

TGFβRII

TGF-β1 receptor II

TNF-α

Tumor necrosis factor-α

VCAM-1

Vascular cell adhesion molecule-1

VSMCs

Vascular smooth muscle cells