Macrophages Under Pressure: The Role of Macrophage Polarization in Hypertension

Abstract

Hypertension is a multi-factorial disease, involving the nervous, renal, and cardiovascular systems. Macrophages are the most abundant and ubiquitous immune cells, placing them in a unique position to serve as key mediators between these components. The polarization of macrophages confers vast phenotypic and functional plasticity, allowing them to act as pro-inflammatory, homeostatic, and anti-inflammatory agents. Key differences between the M1 and M2 phenotypes, the two subsets at the extremes of this polarization spectrum, place macrophages at a juncture to mediate many mechanisms involved in the pathogenesis of hypertension. Neuronal and non-neuronal regulation of the immune system, i.e. the “neuro-immuno” axis, plays an integral role in the polarization of macrophages. In hypertension, the “neuro-immuno” axis results in synchronization of macrophage mobilization from immune cell reservoirs and their chemotaxis, via increased expression of chemoattractants, to end-organs critical in the development of hypertension. This complicated system is largely coordinated by the dichotomous actions of the autonomic neuronal and non-neuronal activation of cholinergic, adrenergic, and neurohormonal receptors on macrophages, leading to their ability to “switch” between phenotypes at sites of active inflammation. Data from experimental models and human studies are in concordance with each other and support a central role for macrophage polarization in the pathogenesis of hypertension.

Introduction

Mononuclear phagocytic immune cells serve as a cohesive cellular component in explaining the immune system’s role in the pathophysiology of hypertension. Essential or primary hypertension is a complex multi-factorial disease that involves the nervous, renal, and cardiovascular systems. Macrophages are the most abundant and most widespread, yet most primitive, immune cells in tissues, including the organ systems involved in hypertension (1). Thus, it is no surprise that to function in diverse environments, macrophages possess an extraordinary degree of plasticity. This plasticity is manifested in a sophisticated framework of functional and phenotypic differentiation, i.e. polarization. At its extremes, this polarization is characterized by the M1 and M2 phenotypes. Although this is an oversimplification of macrophage polarization, identifying key differentiating characteristics between these two macrophage populations can be utilized to begin understanding the complex role of macrophages in the pathophysiology of hypertension. Building a “macrophage-centric model” will help explain the interrelatedness of the neural, renal, and vascular components of hypertension. The most important contribution of this review will be to introduce the “neuro-immuno” axis as an important force in macrophage polarization. Based on the available data, this review will synthesize an interdisciplinary model of macrophage polarization, where the nervous system plays an integral role in synchronizing mobilization of macrophages from immune reservoirs and their chemotaxis to tissues/organ systems involved in the development of hypertension. Finally, we will also review the evidence for the role of inflammation, with a focus on macrophages, in clinical essential hypertension.

Key Aspects of Macrophage Polarization

Macrophages not only play an integral role in the innate immune response, but also carry out homeostatic functions. To carry out these diverse duties, macrophages harbor the ability to polarize and morph into subsets, demonstrating diverse phenotypic and functional plasticity. Although macrophage polarization is deeply intriguing and profound, a complete review of macrophage polarization is beyond the scope of this review. Here we will briefly review macrophage polarization, focusing on important characteristics pertinent to our discussion of hypertension.

M1 vs M2

Following their original discovery as phagocytic cells over 100 years ago by Elie Metchnikoff, the plasticity of macrophages began to become more apparent (2). In an oversimplified model, macrophage polarization can be thought of as consisting of two subsets: M1 and M2. The concept of macrophage polarization was initially borne from the observations that macrophages exposed to interferon-gamma (IFN-γ) expressed a different gene-expression profile than those exposed to interleukin-4 (IL-4) (3, 4). IFN-γ induces the differentiation of M1-macrophages, which are inflammatory and referred to as “classically activated” macrophages; where IL-4 induces the anti-inflammatory M2 macrophages, which are categorized as “alternatively activated” (5).

At the outset, there are a couple main differences between M1 and M2 polarized macrophages that position macrophages to play central roles in hypertension. First, it is important to note that the nomenclature of “M1” and “M2” was derived from the effects that these two macrophage populations have on the differentiation of T-lymphocytes. Specifically, M1 macrophages are known to activate and guide Th1 T-lymphocytes and M2 macrophages are associated with induction of Th2 T-lymphocyte responses (6). Based on the T-lymphocyte associations of macrophage subsets, there is indirect evidence for the role of macrophage polarization in hypertension. In a small study of 45 hypertensive patients, Ji et al demonstrated a significant increase in circulating Th1 and Th17 T-lymphocytes, in contrast to a dramatic decrease in Th2 T-lymphocytes (7). In a RAG KO murine angiotensin II (Ang II) dependent model of hypertension, Guzik et al definitively demonstrated dependence of Ang II hypertension on the presence of T-lymphocytes (8). Similarly, IL-17 has been shown to be integral in the promotion of Ang II mediated hypertension (9). Other studies have also confirmed the critical role of inflammatory T-lymphocytes as essential in the pathophysiology of hypertension (10–13). Second, L-arginine’s metabolic fate, which differentiates the M1 and M2 phenotypes at the molecular level, has potentially important implications for hypertension. M1 macrophages metabolize L-arginine to nitric oxide (NO) via inducible nitric oxide synthase (iNOS or NOS2); where M2 macrophages metabolize L-arginine to ornithine (which promotes tissue growth, repair, and fibrosis) via activation of arginase-1 (14). Discovered in the 1980’s, NO has long been known to play an important role in cardiovascular disease, including hypertension (15). However, NO has a complicated relationship with cardiovascular disease. Not only can NO be synthesized by different tissues and enzymes (neuronal NOS or NOS1, inducible NOS or NOS2, endothelial NOS or NOS3), but its effects are concentration-dependent (16). Abnormalities in NO and endothelial vasodilation have been shown to be abnormal in hypertensive patients (17). Correlating with clinical studies, experimental models of hypertension have also been shown to have abnormalities in NO synthesis and bioavailability (18–20). A major mechanism in the development of essential hypertension appears to be the decreased bioavailability of NO (17) or interference in its regulation of natriuresis (21). Reactive oxygen species (ROS), a product of M1 macrophages, are known to scavenge NO, thereby implicating it as a possible mechanism in limiting NO bioavailability (17). Interestingly, the role of ROS in tilting the balance of polarization towards the M1 or M2 phenotype is less clear. ROS are known to play an important role in skewing the polarization of macrophages towards the inflammatory M1-phenotype as well as recruiting M2-macrophages (22). Despite the fact that ROS are known to be increased in patients with hypertension and experimental models of hypertension, their causative role in essential hypertension has not been shown and remains uncertain (23, 24). Nonetheless, ROS do likely play a key role in the pathophysiology of hypertension, including end-organ damage (25). Finally, macrophages are involved with the process of sodium balance, a cellular activity that has been shown to be related to lymphangiogenesis and blood pressure regulation, discussed further below (26). Thus, at the outset, based on key physiological aspects of macrophage polarization, it appears that there is strong evidence for the role of macrophage polarization as a key mechanism in the development of hypertension. Adding to the complexity is the limited knowledge about macrophage polarization itself.

Macrophage Polarization – Still Developing

Although the dichotomous polarizations of M1 and M2 phenotypes appear straight forward and concrete, it is much more complicated, with new phenotypes and polarizing stimuli (beyond IFN-γ and IL-4) being continuously discovered. For example, similar to IL-4, interleukin-21 (IL-21) has been shown to influence macrophage polarization towards the M2-phenotype (27, 28), through which it may play an important role in the pathogenesis of hypertension and end-organ damage. Similarly, aldosterone has been shown to promote the balance of macrophage polarization in the direction of the M1 inflammatory phenotype (29). Consequently, the picture of a ‘simple phagocytic macrophage’ has morphed into an image of an immune cell that is at the center of a sphere with the potential to follow innumerable radiant pathways of phenotypic and functional diversity. Within just 4 years of their initial description in 2000 (6), the M1–M2 paradigm had already morphed into further subclassification of the M2 macrophage populations into M2a, M2b, M2c, and M2d (5, 30). The complexity of the various macrophage subsets, both known and yet to be discovered, is perceived as being potentially so vast that it has been equated to a color-wheel (31), where slight variations may exist on even known macrophage populations, leading to new phenotypes. Although useful, a color-wheel does not necessarily indicate relatedness between the colors. Here we introduce the phylogenetic tree as a conceptual framework for understanding macrophage polarization, where variations in subsets possess some degree of relatedness to each other (Figure 1). This is exemplified by the various subsets within the M2-phenotype and the, more recently identified, Tumor Associated Macrophages (TAM) (32, 33). Additionally, recent identification of macrophages that express CD169 and the T-cell receptor (TCR), not yet clearly falling into either the M1 or M2 “camps” further complicate matters (34). CD169-positive cells are implicated in patients with renal disease and have been shown to play a role in inducing proteinuria and renal damage (35–37), potentially playing a role in the development of hypertension.

Figure 1. Tree of Macrophage Polarization.

Macrophage polarization has been simplified into the M1 and M2 subsets. However, new markers are continually being discovered and add to the complexity of the paradigm of macrophage polarization. Here we represent a simplified over view of the macrophage subsets, in addition to the M1 and M2 phenotypes, that have been discovered. This includes the tumor assocaited macrophage (TAM), the T-cell receptor-positive (TCR+), CD169-positive, CD161a-positive, and various known subpopulations of the M2 (M2a, M2b, M2c, and M2d). Although new markers are continually being discovered, it is likely that the various macrophage subsets are related to each other and their relatedness will need to be reflected in a hierarch such as this phylogenetic tree.

Identification of macrophage subsets is based on expression of specific extra and intracellular proteins. Traditionally, M1 macrophages are identified by the presence of increased levels of CD68, major histocompatibility complex-II (MHC-II), CD80, CD86, CC chemokine receptor 2 (CCR2, receptor for monocyte chemoattractant protein-1, MCP-1), Ly6, and CD62L on their cell surface. On the other hand, M2 macrophages are phenotyped by increased cell-surface expression of CD163 (scavenger receptor), CX3C chemokine 1 (CX3CR1, the receptor for fractalkine), CD206 (mannose receptor), YM1 and YM2 (members of chitinase and chitinase-like molecules), vascular endothelial growth factor (VEGF), and acidic mammalian chitinase (AMCase) (30–32). In addition, the M1 and M2 macrophages have individual cytokine and chemokine secretion profiles. The “classically activated” M1 phenotype is marked by secretion of pro-inflammatory cytokines that include tumor necrosis factor-alpha (TNF-α) interleukin 1beta (IL-1β), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin-18 (IL-18), and interleukin 23 (IL-23). This cytokine profile specifically and primarily directs the development of the Th1 T-lymphocyte, but is also involved in development of Th17 T-lymphocytes (IL-6 and IL-23), which are involved in secretion of interleukin 17 (IL-17) and implicated in autoimmune diseases. As noted earlier, both of these T-lymphocyte subsets are implicated in the development of hypertension (38). Conversely, the cytokine profile of M2 macrophages, which includes tumor growth factor-beta (TGF-β), induces differentiation and activation of regulatory T-lymphocytes (Tregs). M2 macrophages also secrete interleukin-10 (IL-10), a potent anti-inflammatory cytokine (31). In experimental models of hypertension, not only are Tregs decreased in number, but adoptive transfer of Tregs results in a decrease in blood pressure. These studies demonstrate that the anti-inflammatory actions of these cells may be protective against the development of hypertension and end-organ damage (39–41). As referenced above, the concept of macrophage polarization is relatively new and still evolving with more cellular markers and cytokine profiles remaining to be discovered or, in some cases, rediscovered and reclassified. Thus, the ‘Tree of Macrophage Polarization’ is only a sapling and has much growth to accomplish. This is exemplified by studies in the Spontaneously Hypertensive Rat (SHR), a genetic model of essential hypertension.

Macrophage Polarization in the Spontaneously Hypertensive Rat

CD161a: A New Marker of M1 Macrophages

CD161a, a member of the lectin-like receptor subfamily B member 1 (NKR-P1a), was initially identified as a marker of natural killer (NK) cells (42). However, CD161a is also expressed on antigen presenting cells such as monocytes and macrophages (43). During the late 1990’s, CD161a was noted to be a robust cellular marker of an inflammatory macrophage phenotype involved in acute kidney allograft rejection in rats (44). Lectin-Like-Transcript-1 (LLT1) has been identified as a ligand for CD161a and this interaction may play a key role in immunomodulatory functions of Th17 cells (45). Our group was the first to identify an abnormal predominance of CD161a-positve immune cells in young (3–5 weeks old) SHR prior to the elevation of blood pressure (46), suggesting that this immune cell population may play a causative role in the development of hypertension. Later, we showed that this immune cell population differentiated into CD68-positive M1-macrophages following activation of nicotinic cholinergic receptors (nAChR) (Figure 2a) and that monocyte chemoattractant protein (MCP-1) and LLT1 were over-expressed in the kidney of the SHR, suggesting a mechanism for homing of these cells (47). Cholinergic stimulation also led to the infiltration of these CD161a+/CD68+ M1 macrophages into the renal medulla and subsequent development of hypertension in young pre-hypertensive SHR (47), implicating renal inflammation by these cells as a causative mechanism in the development of hypertension (a process we refer to as “cholinergic-mediated hypertension”) (47). In support of these findings, treatment with a novel therapeutic agent inhibited the induction of iNOS and M1 macrophage activation, via increased expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ), and led to normalization of blood pressure in the SHR (48). This is in line with the known effects of PPAR-γ to polarize macrophages towards the M2-phenotype (49). Normalizing blood pressure via the inhibition of M1 macrophage activation is a key factor in demonstrating the role of macrophage polarization in hypertension (loss of macrophage ‘inflammatory potential’). To strengthen the causative link between macrophage polarization and hypertension, based on Hill’s criteria, it is also important to show that induction or activation of the anti-inflammatory M2 macrophage retards or normalizes elevations in blood pressure (gain of macrophage ‘Anti-inflammatory potential’).

Figure 2. Imbalance of M1 and M2 macrophage populations in SHR.

A) Splenocytes from young (3–5 week old) pre-hypertensive Spontaneously Hypertensive Rats (SHR) and their age-matched Wistar Kyoto (WKY) controls were isolated and cultured in the absence (media alone) or presence of nicotine (10 micromolar) in vitro for 48 hours. Splenocytes were then harvested and stained with antibodies against CD161a and CD68 (ED1) and underwent flow cytometry. Nicotine induced a significant increase in the CD161a+/CD68+ M1 macrophage population, compared to the WKY; B) Splenocytes were isolated from young SHR and WKY following an 2-week in vivo infusion of nicotine bitartrate (15mg/kg/day) and stained for CD4 and CD163, a marker of the M2 macrophage. There was over a 50% reduction in the nicotine-infused SHR, compared to WKY.

The M1/M2 Ratio

It appears that the ratio of M1/M2 macrophage plays an integral role in the pathophysiology of hypertension. Ndisang et al. have documented over a 50% decrease in the M2 macrophage markers CD163 and CD206 by Western blot of hepatic homogenates in the SHR and demonstrate that induction of the M2-macrophage population, thereby decreasing the M1/M2 ratio, results in normalization of blood pressure in adult SHR with established hypertension (Figure 3) (50). Using flow cytometry, we characterized the CD163 positive M2 macrophage population as CD4^Lo in the pre-hypertensive SHR and also find an over 50% reduction, compared to the age-matched control (Wistar Kyoto, WKY) following the induction of ‘cholinergic-mediated hypertension’ (Figure 2b). In addition, Gomolak et al. demonstrated a dose-response effect between angiotensin-II (Ang II) infusion, the induction of systolic hypertension, plasma IL-6 levels, vascular infiltration with CD68-positive M1 macrophages, and endothelial dysfunction (51). Beginning at a ‘low-intermediate’ dose (200ng/kg/min) of Ang II, there was a subacute rise in systolic pressure with a trend towards increased vascular M1-macrophage infiltration, and endothelial dysfunction. These changes were more pronounced at a ‘high-intermediate’ (400ng/kg/min) and “high” (1000ng/kg/min) dose of Ang II (51). Utilizing a murine model with inducible diphtheria toxin receptor, Wenzel et al. elegantly established that Ang II-induced hypertension is dependent on the presence of CD11b+/F4-80+ M1-macrophages (52). Additionally, deficiency of m-CSF (monocyte colony stimulating factor) results in selective monocytopenia and resistance to Ang II dependent vascular injury, oxidative stress, and hypertension (53–56). Although, the increased M1/M2 ratio appears to be a critical component in the development and maintenance of hypertension, the native causative factor/s responsible for this imbalance in hypertension have not been clearly identified. One known factor in the induction of the M1-phenotype is activation of innate immune receptors (aka ‘pathogen recognition receptors’, PRR).

Figure 3. Induction of M2 Macrophages Leads to Decrease in the M1/M2 Ratio and Decrease in Blood Pressure.

Data from Ndisang and Mishra 2013 (46) redrawn to demonstrate the decrease in blood pressure (■) (mmHg) following induction of the M2-macrophage ( ) and the decrease in the M1-macrophage ( )(with consequent decrease in the M1/M2 ratio) in the adult Spontaneously Hypertensive Rat with established hypertension.

Toll-like Receptors and Macrophage Polarization

Toll-like receptors (TLR) are innate immune receptors that detect “pathogen-associated molecular patterns” (PAMPs) and “danger associated molecular patterns” (DAMPs). Macrophages are known to express a wide range of TLR (57). Activation of TLR skews the polarization of macrophages to the M1 phenotype (58), potentially, playing an important role in essential hypertension by altering the M1/M2 ratio. To explain the pro-inflammatory milieu and polarization of macrophages towards the M1 phenotype in hypertension, some have postulated the presence of “neoantigens” that trigger an inflammatory innate immune response (59). To date, however, the identity of these “neoantigens” remains elusive. Nevertheless, it is known that extraneous factors can regulate TLR expression and activation, skewing macrophage polarization towards the M1 phenotype. To this effect, we demonstrated that activation of nAChR not only induces the expansion of a CD161a+/CD68+ M1 macrophage population, but also modulates the innate immune response, leading to an exaggeration of the pro-inflammatory cytokine secretion in response to TLR stimuli from young pre-hypertensive SHR derived immune cells. This was in stark contrast to the observed anti-inflammatory cholinergic effect following activation of TLR in age-matched WKY-derived immune cells (60). Interestingly, the immune response to TLR activation of immune cells derived from the young SHR and WKY were similar in the absence of pre-exposure to cholinergic stimuli (46). These findings confirm the presence of a previously described “cholinergic anti-inflammatory” effect (61, 62) and demonstrate the presence of an paradoxically abnormal “cholinergic mediated pro-inflammatory effect” in the pre-hypertensive SHR. This abnormal pro-inflammatory regulation of macrophage polarization by cholinergic receptors represents a novel key mechanism in macrophage polarization and the development of hypertension. More importantly, it establishes precedence for the regulation of immune responses, including macrophage polarization, by engagement of cholinergic, adrenergic, and neurohormonal receptors (e.g. angiotensin type 1 receptor, AT1R and the mineralocorticoid receptor) directly on immune cells.

The “Neuro-Immuno” Axis in Hypertension: a new force in Macrophage Polarization

An important force in the polarization of macrophages is the interaction between the nervous and immune system, i.e. the “neuro-immuno” axis. Specifically, the “neuro-immuno” axis refers to the bidirectional communication between the nervous and immune system. In the forward direction, central nervous system (CNS) activity directly regulates immune cell function and differentiation through efferent nerve fibers; while, in the reverse direction, immune cells/cytokines directly impact the activation state of the CNS via afferent neural pathways. To complicate matters, neural regulation of immune cells can occur via neuronal (direct) and non-neuronal (indirect) mechanisms. Neuronal regulation refers to the direct effects of the CNS nerve fibers on immune cells; where non-neuronal regulation refers to the effects of neurotransmitters and neurohormonal agents secreted from non-neuronal cells and acting on the immune cells in an autocrine or paracrine manner.

CNS to Immune

Neuronal Regulation

The autonomic nervous system has long been known to directly innervate immune cell compartments (63), where nerve fibers make close contact with immune cells at the “neuro-immune synapse” (64). Studies have demonstrated evidence for autonomic neuronal regulation of immune responses (61, 65), leading to a streamlined model of the “neuro-immuno” axis, where the parasympathetic nervous system (PNS) mediates anti-inflammatory and the sympathetic nervous system (SNS) mediates inflammatory immune responses (Figure 4a). Borovikova et al. had initially demonstrated that stimulation of the vagus nerve (PNS) exerts an inhibitory influence on endotoxin induced M1 macrophage secretion of pro-inflammatory cytokines (61), referred to as the “cholinergic anti-inflammatory reflex”. The anti-inflammatory effect of the vagus nerve was found to be meditated by the alpha7-nicotinic acetylcholine receptor (α7-nAChR) (62). Vagal nerve stimulation also induces the activation of the Jak2-Stat3 transcription factors, which are known to polarize macrophages to the M2 phenotype (66, 67). In contrast, induction of splenic sympathetic nerve activity, in response to infusion of Ang II into the CNS, has been shown to lead to an increase in mRNA of M1 macrophage pro-inflammatory cytokines in the spleen (68). Carnevale et al, has also demonstrated that sympathetic innervation of the spleen is required for the development of hypertension in response to infusion of Ang II, definitively demonstrating the integral role of Ang II’s “neuro-immuno” effects in the development of Ang II-dependent hypertension. Splenic sympathetic innervation appears to mediate communication between CD68-positive M1 macrophages and T-lymphocytes, leading to egression of the T-lymphocytes, end-organ damage, and the development of hypertension (69, 70). More recently, in patients, increased renal sympathetic nerve activity was shown to be involved in macrophage activation (65). Conversely, renal denervation results in a significant decrease of F4/80-positive inflammatory M1 macrophages into the renal medulla and increase in the expression of kruppel-like factor-4 (klf4) (71), a factor that induces polarization of macrophages to the M2 phenotype (72). Hence, based on these studies one may conclude that direct activity of the PNS favors the M2 phenotype and SNS favors the M1 phenotype (Figure 4b). Leaving the overview of the neuronal “neuro-immuno” axis, it appears the direct neuronal effects of the PNS and SNS on the immune system are discrete and straightforward. Unfortunately, the lines between the effects of activating adrenergic (sympathetic) and cholinergic (parasympathetic) receptors directly on immune cells in an autocrine or paracrine fashion, i.e. non-neuronal regulation, are less crisp.

Figure 4.

Figure 4A. Overview of the “Neuro-Immuno” Axis in Hypertension.

The autonomic nervous system is comprised of the sympathetic/adrenergic and parasympathetic/cholinerigic arms. Activation of the adrenergic component leads to direct pro-inflammatory influence; where the cholinergic neural arm mediates an anti-inflammatory response on macrophages and other innate immune cells. Pro-inflammatory innate immune regulation leads to an pro-inflammatory adaptive immune (T- and B-lymphocytes) response and the development of hypertension, marked by end-organ inflammation.

Figure 4B. The Neuronal “Neuro-Immuno” Axis in Macrophage Polarization.

Activation of the sympathetic autonomic nervous system mediates polarization of macrophages towards the M1 phenotype. In contrast, activation of the parasympathetic autonomic nervous system mediates polarization of macrophages towards the M2 phenotype.

Non-Neuronal Regulation

It is well known that macrophages express cholinergic and adrenergic receptors (63). Although direct neuronal regulation of immune cells via the SNS mediates inflammatory immune responses consistent with activation of M1 macrophages, activation of adrenergic receptors on macrophages is slightly more complicated. Activation of alpha-1 and alpha-2 adrenergic receptors seems to induce polarization towards the inflammatory M1 phenotype (73); whereas activation of beta-2 adrenergic receptors mediates polarization towards the anti-inflammatory M2 phenotype (74). Less is known about the effects of cholinergic receptors on macrophages, with the exception of the well-investigated alpha7-nAChR (α7-nAChR), which has been shown repeatedly to exert anti-inflammatory effects, skewing the innate immune response towards the M2 phenotype (75). Activation of the α7-nAChR has not been demonstrated to decrease blood pressure or prevent/delay the onset of hypertension, but it has been demonstrated to inhibit end-organ damage in experimental models of hypertension (76). However, although the α7-nAChR exerts anti-inflammatory effects, cholinergic receptors are a diverse group of receptors. Following the template of the adrenergic receptors, it is clear that while some nAChR exert anti-inflammatory effects (α7-nAChR), others harbor the ability to elicit pro-inflammatory immune responses (77, 78). To this effect, our laboratory first demonstrated that activation of nAChR ex vivo and in vivo leads to the selective induction of M1 macrophages in the young pre-hypertensive SHR, but not in the age-matched WKY (47), corresponding with the premature development of hypertension. Investigating the effects of cholinergic receptors on innate immune function, much less on macrophage polarization, is a new frontier. As it stands, the current database of knowledge regarding nAChR is based on studies of nerve cells and muscle tissue, which serves as the bedrock of our understanding of nAChR; hence the categories of neuronal nAChR and muscle-type nAChR (79). Studies investigating the expression of nAChR in immune cells have primarily been limited to the α4, α7, and β2-nAChR (80). Thus, there is a large gap in our knowledge of the repertoire of cholinergic receptors in immune cells. Currently, our laboratory is actively working to characterize nAChR expression in immune cells, in an attempt to identify the nAChR/s involved in mediating the pro-inflammatory M1 differentiation of the pro-hypertensive CD161a-positive macrophages.

Immune to CNS

Generally, the nervous system is considered an immune-privileged site, separated from the circulating immune cells and cytokines/chemokines by the blood-brain barrier (BBB). In thinking about the interfaces of the CNS with the peripheral immune cells as cytokines, it is important to identify 3 main interfaces: the blood-brain barrier (BBB), choroid plexus (CP), and the circumventricular organs (CVO) (Figure 5). These interfaces are presented in order of increasing “leakiness” and contact with the systemic circulation. The BBB is the most impenetrable to circulating cytokines, neurohormonal factors, and immune cells; where the CVO are penetrated by fenestrated capillaries and are considered to have a high level of permeability, allowing direct contact with systemic factors and cells (81). The CP is a barrier between the systemic circulation and the brain that lines the ventricular compartment in vertebrates and is composed of a combination of endothelial and epithelial cells; although other immune cells are also known to be present, including macrophages. The primary function of the CP is to generate cerebrospinal fluid for the CNS, which it does through a close interaction with fenestrated capillaries blood vessels (82). The CVO is comprised of the median eminence, neurohypophysis, organum vasculosum lamina terminalis (OVLT), the subfornical organs (SFO), and area postrema (AP). In addition, there are areas within the CNS (the forebrain and hindbrain) that are in very close proximity to the systemic circulating factors and immune cells. Important structures in these areas are the paraventricular nucleus (PVN), rostroventrolateral medulla (RVLM), anteroventral third ventricle (AV3V), and nucleus tractus solitarius (NTS) (Figure 5). A comprehensive review of neurobiology is beyond the scope of this review, but a simplified overview is presented. In essence, the NTS and PVN are areas for ‘input’ from the CVO and afferent nerve fibers. Neuronal projections from these areas then communicate with other areas of the CNS, such as the RVLM, which is the primary center for sympathetic outflow from the CNS. Key to implicating these centers in the development of immune-mediated hypertension was a study by Shi et al. revealing the pro-hypertensive effects of IL-1β delivered directly to the PVN or into the ventricular space of rats (83).

Figure 5. Central Nervous System Interfaces and Hindbrain/Forebrain Neural Centers Involved in the Regulation of Blood Pressure.

Schematic depicts the Blood-Brain Barrier (BBB) and Choroid Plexus (CP) along with the associated structural (astrocytes and pericytes) and immune cells. Immune cells include microglia, perivascular/infiltrating macrophages, dendritic cells (DC), monocytes, and macrophages. Hindbrain and forebrain structures include the circumventricular organs (CVO), neurohypophysis (pituitary & hypothalamus), organum vasculosum lamina terminalis (OVLT), subfornical organs (SFO), area postrema (AP), paraventricular nucleus (PVN), rostroventrolateral medulla (RVLM), anteroventral third ventricle (AV3V), subcommisural organ (SCO), and nucleus tractus solitarius (NTS).

With the basic view presented above, one can begin to understand the communication of the immune system with the CNS. Pro-inflammatory cytokines, that are generated in the periphery as a result of immune responses to antigens (self or foreign), such as IL-1β, IL-6, and TNF-α, are pivotal in this communication (84, 85). These cytokines can communicate directly with the CVO or induce alterations in the in the adherens and tight junctions of the BBB and induce increased permeability to cytokines to gain access to the CNS (86, 87). Cytokines at local sites of inflammation also have the ability to trigger afferent nociceptive (somatic) and vagal fibers in the periphery, thereby communicating with the NTS and PVN via neuronal pathways (84), potentially activating increasing sympathetic outflow from the CNS and leading to hypertension. Thus, inflammatory M1 macrophages at localized sites of inflammation in the periphery can signal to the CNS to raise blood pressure transiently through inflammatory cytokines (which would be protective in acute illness); however, in the chronic inflammatory state, this signal may trigger an abnormal pro-inflammatory immune response within the CNS, leading to essential hypertension.

CNS Macrophages

Although there are a variety of immune cells within the CNS, we will focus on those cells relevant to the discussion of macrophage polarization, only briefly mentioning other cells as needed. It is most useful to think of immune cells as 1) outside the CNS, 2) at the interfaces of the CNS with the periphery, and 3) within the CNS. The role of macrophages and immune cells outside the CNS has been discussed elsewhere in this review. Here we will focus on how macrophages and their actions at the interfaces and within the CNS can regulate hypertension. The BBB is a complicated structure primarily composed of processes of astrocytes with close proximity to penetrating blood vessels in the brain parenchyma. Astrocytes are the most numerous non-neuronal cells in the CNS and play an important structural role in the maintenance of the BBB, but have also been found to have immune functions (marked by the secretion of IL-6) (88). Perivascular macrophages (PVM) are an important mononuclear phagocytic cell that also surround the blood vessel and are just outside the BBB, able to communicate with astrocytes (89). TGF-β, a hallmark of the M2- phenotype, is secreted by multiple cells at the BBB in an attempt to maintain its integrity (89). Thus, it is not surprising that PVM primarily express CD163, a M2-phenotype marker (90). PVM surround the penetrating blood vessels in the brain parenchyma and are primarily replenished by blood-derived macrophages (91). Despite their native, anti-inflammatory state, the PVM can be polarized towards an M1 phenotype by known pro-M1 stimuli, such as endotoxin (systemic or local). Once activated, PVM signal to neighboring immune cells, endothelial cells, and parenchymal microglia through pro-inflammatory cytokines such as IL-1β (92). Activation of PVM leads to induction of ROS and is implicated in damaging neurocognitive and vascular effects of hypertension (93). Consistent with this finding, Liu et al found an approximately 50–100% increase in the number of PVM present in adult hypertensive SHR and stroke prone SHR, compared to the age-matched WKY (94). Similarly, macrophages are also present in the stroma of the CP and their activation to the M1-phenotype in hypertension has also been implicated in cognitive dysfunction (95). Macrophages in the stroma of the choroid plexus (CP-macrophages) can transmigrate across the choroid plexus and line the ventricular boundary of the CNS (96). CP-macrophages are also derived from systemic blood-derived macrophages, but their roles in hypertension development/maintenance or hypertensive cerebral dysfunction have not been studied in-depth. Nonetheless, due to their relationship with blood derived macrophages they can polarize to M1 or M2 macrophages and secrete cytokines directly into the CSF, inducing M1 polarization in parenchymal microglia (discussed below). Summarizing the role of macrophages at the BBB and CP, it appears activation to the M1-phenotype primarily leads to transduction of hypertensive damage to the CNS, leading to neurocognitive dysfunction. However, microglia play a much more important role in the development of neuroinflammation and hypertension.

Neuroinflammation is known to play a significant role in the development of essential hypertension (97). The concept of neurogenic hypertension has traditionally revolved around the increased sympathetic outflow of the CNS to the periphery, leading to increased vascular resistance and end-organ damage (98–100). Microglia are resident macrophages within the brain parenchyma. Microglia are derived from the embryonic yolk sac prior to the ninth embryonic day in rodents (101), proliferating and maintaining homeostasis within the CNS. In contrast to PVM and CP-macrophages, microglia have little to no dependence on systemic circulating monocytic/macrophage precursors (102). It has become evident in the past several years that microglial activation, i.e. neuroinflammation, leads to the secretion of inflammatory cytokines that are sympathoexcitatory, linking immune activation to increased sympathetic outflow from the CNS, a key mechanism in pathogenesis of essential hypertension (83, 103–107). Similar to blood-derived macrophages, microglia can polarize towards an M1 or M2 phenotype (58, 108). Although microglia can be influenced to polarize towards an M1 phenotype in response to cytokine signals transduced through immune cells at the interfaces of the CNS (PVM, CP-macrophages, and T-lymphocytes), other factors also directly activate the microglia. At this point, the importance of the renin-angiotensin-aldosterone system (RAAS) becomes a central and critical component of neuroinflammation and neurogenic hypertension.

Ang II is a key component of RAAS that results in elevated blood pressure via vasoconstriction, increased sodium reabsorption, as well as sympathomimetic effects on the CNS. The mechanism for the CNS effects of Ang II has been an area of active investigation. Recently, Ang II was shown to activate microglia, polarizing them towards an M1-phenotype, inducing the development of hypertension (83). Minocycline, an anti-inflammatory antibiotic, was shown to abrogate hypertension in this model by reversing the polarization of the microglial towards an M2-phenotype as marked by an increase in IL-10, and decrease in the pro-inflammatory M1 cytokines (IL-1β, IL-6, and TNF-α) (83). In 2015, Shen et al., utilized a CD11b-DTR transgenic mouse model to selectively deplete microglia in the motor cortex and PVN, resulting in resolution of hypertension in an Ang II dependent model, confirming the role of M1-polarized microglia in neurogenic hypertension (106). This is consistent with earlier findings of an anti-inflammatory effect of angiotensin II type I receptor (AT1R) antagonists in microglia exposed to endotoxin (109). Vascular inflammation in the brainstem has been proposed as a potential explanation in the SHR for the development of hypertension, possibly through decreased oxygen delivery to the CNS (110). Interestingly, cerebral ischemia leads to upregulation of microglial expression of Ang II, AT1R, and AT2R (angiotensin type 2 receptor) in microglia, establishing a role for the autocrine and paracrine inflammatory action of Ang II in the CNS (111). Of note, Ang II’s induction of inflammatory effects in microglia has been shown to be mediated by the presence of functional TLR4, demonstrating cross talk between the AT1R and TLR pathways (112). Similarly, we had shown that Ang II primed immune cells from young pre-hypertensive SHR in vitro and in vivo to exert an exaggerated pro-inflammatory immune response to TLR activation (46). Taken together, it is clear that Ang II and innate immune receptors work together to shift the balance of microglial activation towards an M1 phenotype, leading to the development and propagation (in a feedforward manner) of essential hypertension. Increased microglial activation and polarization may also result from various inflammatory cytokine input from the CNS interfaces as well. Moreover, polarization towards the M1 phenotype of macrophages, including microglia, within the CNS likely plays a key role in the neurocognitive dysfunction associated with hypertension (93, 94, 113).

RAAS, Inflammation & Macrophage Polarization

At face value, the RAAS system also plays dual contradictory roles in hypertension with regards to inflammation. Systemic Ang II infusion is known to induce the expression of MCP-1 in vascular smooth muscle cells and the kidney (114–116). As a consequence, it is no surprise that, both, the vascular and renal compartments are marked by increased infiltration of inflammatory M1 macrophages with Ang II infusion, along with the corresponding inflammatory cytokine milieu (117–120). From this, it logically follows that MCP-1 deficiency would be protective against vascular inflammation and infiltration of macrophages (117, 118). Interestingly, recent studies have revealed a dynamic component of macrophage biology in the context of renal and vascular inflammation. Specifically, although inflammatory M1-phenotype macrophages initially infiltrate these structures, these cells can ‘switch’ to the M2-phenotype. Confirming the Ang II induced infiltration of the aorta with M1-phenotype Ly6C^hi macrophages, Moore et al. showed that at 7–14 days, these cells began to express the M2 phenotype, marked by increased CD206 and arginase-I (121). Further they showed that antagonism of MCP-1, prevented the macrophage infiltration and subsequent fibrosis associated with Ang II infusion, the latter of which is most likely mediated by the M2 macrophages (121). Similarly, Aki et al. demonstrated the ability of systemic AT1R blockade with olmesartan to induce a switch from an M1 macrophage profile to an M2 phenotype, along with an increase in IL-4, IL-13, and IL-10 (an M2 anti-inflammatory cytokine profile) (122). From these studies, it is important to note that M1 macrophages infiltrate vessel walls and renal interstitium in response to elevated levels of systemic Ang II, which crosses the BBB (123). Moreover, the plasticity of macrophages in their phenotypic and functional diversity is dynamic, with the fluent ability to transition between phenotypes. Despite this, there appears to be a disconnect with studies looking at the direct effects of AT1R engagement on the surface of immune cells.

Immune cells are known to express components of RAAS, including AT1R (124, 125). It appears that the direct engagement of the AT1R on immune cells mediates the polarization of macrophages in the direction of the anti-inflammatory M2-phenotype (126, 127). Kato et al had investigated the effects of selective deletion of AT1R in mice expressing human renin and angiotensinogen genes (chronic RAAS activation) and found that there was a preserved hypertensive response, increased renal fibrosis, and increased severity of atherosclerosis (128). Similarly, Crowley et al demonstrated that Ang II infusion in bone marrow chimeric mice with deletion of the AT1R in hematopoietic cells have elevated blood pressures, albuminuria, as well as increased T-lymphocyte and macrophage infiltration into the kidneys (129). In line with these basic science studies, Merino et al demonstrated that the use of losartan, an angiotensin receptor blocker (ARB), in patients led to a significant decrease in the M2 macrophage (CD16-positive) population (130). However, studies demonstrate that renal denervation decreases the expression of MCP-1 and protects against renal fibrosis in mice (131), decreased infiltration of F4/80 macrophages in chronic Ang II infusion (71), and decreased inflammatory macrophages [Harwani unpublished data].

The “neuro-immuno” axis likely explains these seemingly discordant findings. Increased sympathetic drive leads to the increased mobilization of mononuclear phagocytic cells from immune cell reservoirs (70) and simultaneously increases the expression of chemoattractant signals, e.g. MCP-1, in the end-organs, such as the kidney. Simultaneous sympathetic-mediated macrophage mobilization and tissue chemoattractant expression results in an increased influx of M1 Inflammatory macrophages into endo organ tissues. Within these tissues engagement of innate immune receptors, e.g. TLR, with “neoantigens”, activates the M1 macrophages. As the inflammatory cascade ensues, these macrophages encounter activation of AT1R on their cell surface through local Ang II in the tissue (they may even secrete Ang II, and undergo activation in an autocrine/paracrine fashion), which then triggers a gradual transformation to the M2 phenotype, with its consequences of fibrosis etc…(Figure 6).

Figure 6. Role of the “Neuro-Immuno” and Neurohormonal Axis as a force in macrophage polarization in the Development of Hypertension.

Angiotensin II (Ang II) activates the microglia through the angiotensin type 1 receptor (AT1R) and promotes their polarization towards an M1 phenotype. Minocycline is a known inhibitor of Ang II mediated M1 polarization of microglia. M1 polarization of the microglia leads to activation of the sympathetic outflow from the central nervous system (CNS). Increased sympathetic outflow leads to 1) Increased expression of chemoattractant molecules (e.g. monocyte chemoattractant protein-1, MCP-1) in end-organs, such as the kidney. Simultaneously, increased sympathetic outflow mobilizes macrophages from immune cell reservoirs (e.g. spleen), while priming them for polarization towards an M1 phenotype. In response to increased expression of the chemoattractants in the kidney, macrophages infiltrate the renal medulla and are activated to polarize into the M1 phenotype. Renal and vascular inflammation leads to the development of hypertension. As hypertension develops, engagement of the AT1R directly on the surface of the macrophage results in M1 polarized macrohages “switching” into M2 macrophages, responsible for renal/vascular fibrosis and remodeling.

Relationship between Salt and Macrophages Polarization

Polarization of macrophages is also an important component in sodium balance, which has been an important mechanism in the development of hypertension clinically and in experimental models for decades. Osmotic forces due to the presence of sodium are believed to play an important role in the pathogenesis of essential hypertension. By tightly regulating total body sodium, the body attempts to maintain a steady-state to counter the pro-hypertensive effect of sodium. There is no doubt that renal mechanisms play an important role in this process and the renal infiltration of macrophages and inflammation are important in the development of hypertension, but it is also clear that macrophages play an important role in the regulation of dermal interstitial sodium. Dermal interstitial sodium stores were shown to correlate with the induction of hypertension, which was countered by macrophage induced lymphangiogenesis, mediated by the expression of VEGF-C. The expression of VEGF-C was dependent on the expression of tonicity-enhancer binding protein (TONEBP/NFAT5) (132, 133). Increased dietary sodium has also been shown to be a key factor in the M1 macrophage dependent development of Th17 T-lymphocyte mediated autoimmune disease (134, 135). Hence, it is no surprise that TLR induction of iNOS, a hallmark of M1 macrophages, is dependent on expression of TONEBP (136), since NO has the potential to interfere with natriuresis (21). Likewise, TONEBP inhibits IL-10 mediated polarization towards the M2-phenotype (137), supporting the pro-M1 effects of sodium and its regulatory mechanisms.

Evidence Suggesting a Role for Macrophage Polarization in Clinical Essential Hypertension

Multiple organ systems have been implicated in the development of hypertension and pharmacological therapies target these various systems. Despite this, approximately 12% of treated patients remain hypertensive and are categorized as being “treatment resistant” or “refractory to medical therapy” (138). Of those that have reached adequate control of their blood pressure, many of them are on multiple agents. This would seem to suggest that although patients are responding to agents targeting multiple organ systems and pathways, our current therapies are missing an essential core mechanism in the pathophysiology of hypertension: the immune system.

Human data is less abundant than experimental models, but the data that is available supports a role for inflammation and macrophage polarization in essential hypertension, as well as cardiovascular disease. Inflammation has been demonstrated in patients with hypertension by the presence of increased circulating levels of inflammatory cytokines and immune cells in various organ systems (renal, vascular, and neural) that are implicated in the development and maintenance of essential hypertension.

The majority of studies have correlated the presence of hypertension with elevated levels of circulating inflammatory markers, cytokines, and antibodies. Increased levels of IgG immunoglobulins were noted in patients with hypertension (139–141). Hypertensive patients are known to have an increased level of IL-6, IL-1β, IL-1α, IL-18, IL-2, IL-8, TNF-α, IFN-γ, C-reactive protein (CRP), and MCP-1. In contrast, anti-inflammatory cytokines such as IL-10 have been noted to be decreased in patients with hypertension (142). Studies have also noted the presence of agonistic autoantibodies to the angiotensin type I receptor (AT1R) as well as the presence of host derived antigens that activate innate immune responses (143–145). In 2007, Sesso et al. attempted to demonstrate a temporal relationship between the presence of inflammatory cytokines and the risk of developing essential hypertension in a nested case-control study of 400 women, showing that IL-6 and CRP were elevated prior to the development of the hypertension (146). The results showed a strong association between the elevated presence of CRP and the development of hypertension, but not IL-6. Similarly, elevated levels of inflammatory cytokines (CRP, IL-6, and soluble ICAM-1) were associated with the development of hypertension in an analysis of men in the Physician Health Study in another prospective study (147). Although the presence of cytokines have been an important initial step in establishing inflammation as a possible causative factor in the development of hypertension, the presence and lack of immune cells have been helpful in shedding light on the mechanisms.

Both the innate and adaptive immune cells have been implicated in the development of hypertension. The immunological paradigm is that immune responses begin with activation of the innate immune system, which then goes on to activate the adaptive immune arm through presentation of antigens and secretion of pro-inflammatory cytokines. In the 1950’s investigators demonstrated a correlation between renal lymphocyte infiltration and scarring in hypertensive patients undergoing sympathectomy (148, 149). Another study demonstrated that severity of hypertension was independent of race and closely associated with the presence of CD68-positive M1 inflammatory macrophages in the kidney (150). Vascular inflammation has been suggested to lead to vascular “stiffness” based on the correlation of increased CRP, TNF-α, IL-6, and adiponectin in blood vessels with increased pulse-wave velocity, a measurement of vascular stiffness that corresponds with hypertension (151–153). These studies are supported by evidence that peripheral blood monocytes (PBMCs) isolated from hypertensive patients are preactivated based on the increased secretion of IL-1β when cultured with Ang II and lipopolysaccharide (LPS), M1 polarizing stimuli (154). Interestingly, clinical inhibition of IL-1β in patients over 4 years was shown to decrease cardiovascular events in patients independent of their cholesterol levels, providing evidence for the role of the innate immune response in cardiovascular disease (155). In hypertensive patients, CD8-positive, but not CD4-positive T-lymphocytes are increased in the circulation of (156), serving as indirect evidence for a Th1 mediated immune response (which is mediated by M1 macrophages). Overall, the inflammatory state in patient studies early on supports a role for the M1/Th1 mediated pro-inflammatory immune response. However, since that time, we have more recent studies that have looked at the differentiation of macrophages by subsets in hypertensive patients.

In humans, macrophages subsets are grouped by the expression of CD14 and CD16 based on a nomenclature system approved by the Nomenclature Committee of the International Union of Immunologic Societies. This system breaks the macrophage populations into three subsets: CD14⁺⁺/CD16⁻⁻ (“Classical Monocytes/Macrophages”), CD14 ⁺/CD16⁺ or CD14 ⁺⁺/CD16⁺ (“Intermediate Monocytes/Macrophages”), andCD14^{dim or +}/CD16⁺⁺ (“Non-classical Monocytes/Macrophages”) (157). In essence, one can simplify these into two primary groups of macrophages: CD14-positive and CD16-positive, which can be taken to be correlates of the M1 and M2 phenotypes, respectively. Interestingly, similar to the effects seen in the SHR, activation of PPAR-γ decreased the expression of CD14, inhibiting the polarization of macrophages towards the M1 phenotype in diabetic patients (158). This CD14-positive vs CD16-positive nomenclature has been helpful in identifying polarization of macrophages in clinical hypertension and serving as a marker for morbidity and mortality in hypertensive patients with cardiovascular disease. A family of molecules referred to as angiopoietin and their receptor Tie-2 have been implicated in patients with hypertension (159) and are involved in the vascular ‘stiffness’, remodeling, and angiogenesis of blood vessels (160). Marketou et al. demonstrated that CD14+ M1 phenotype macrophages were present in the blood vessels of patients with hypertension and express angiopoietin and their receptor, Tie-2 (161). Re-invoking the M1/M2 ratio, the presence of circulating CD14-positive M1 macrophages was found to positively correlate with the incidence of cardiovascular events; where the presence of CD16-positive M2 macrophages negatively correlated with the development of cardiovascular events, suggesting a protective mechanism of a decreased M1/M2 ratio (162). Directly implicating CD14-positive human macrophages in the development of clinical hypertension is their robust expression of angiotensin converting enzyme (ACE) (163), demonstrating their potential involvement in hypertension through participation in the RAAS, which may lead to feedforward activation of monocytes and “switching” between theM1 and M2 phenotypes. This theory is consistent with the finding that monocytes isolated from hypertensive patients are preactivated based on their increased secretion of IL-1β when cultured with Ang II and LPS (154). Supporting the theory of the “Neuro-Immuno” axis as a force in macrophage polarization and participation in RAAS and the development of hypertension, the presence of CD14-positive macrophages in the kidney directly correlated with the severity of renal damage in patients with chronic kidney disease (130). In addition to their activation state, the ability of macrophages from patients with elevated blood pressure to mobilize was increased and correlated with systemic levels of TNF-α (164).

Translational Significance

The translational significance of the role of macrophage polarization in hypertension is multi-faceted. Thus far, our current therapies target hypertension from a multitude of directions: vasodilators, diuretics, angiotensin converting enzyme (ACE) inhibitors, Ang II receptor blockers (ARBs), central sympatholytic alpha receptor agonists, and aldosterone antagonists. The majority of these agents target the RAAS, which appears to play an important role in the modulation of the M1/M2 ratio. New therapies that target the signaling cascade of the M1 and M2 macrophage, such as nAChR and intracellular signaling pathways of the RAAS, may harbor a new frontier in combating hypertension and helping us understand the benefit or lack of benefit on morbidity/mortality of current and future therapies. For example, the beneficial effects of aldosterone antagonism in hypertension and cardiovascular disease may be explained by its ability to induce a “switch” from the M1 to the M2 phenotype, limiting the amount of tissue inflammation. Another example is the development of hypertension in patients being treated with anti-VEGF therapies for malignancies. Knowing that M2 macrophages express VEGF, we may begin to understand the mechanisms responsible for the pro-hypertensive effects of these therapies. More importantly, currently therapies for hypertension such as renal denervation may be able to modulate other inflammatory disease processes. More broadly, neural denervation and/or stimulation may serve as important therapies in inflammatory disorders, including hypertension, if we can determine which neural pathways can modulate macrophage polarization so as to limit inflammation. Based on the model proposed here, we would hypothesize that these therapies will have differential effects based on the timing of their implementation in the disease process, i.e. they may be more beneficial earlier than later in the disease process.

Closing Thoughts

Macrophages are ubiquitous and universally present in all tissues. This includes the organ systems involved in essential hypertension: nervous, renal, and cardiovascular. Our understanding of macrophage biology and its role in hypertension, as well as other disease processes, is quickly advancing. The ability of macrophages to polarize into numerous phenotypes with functional versatility places them in the unique position to serve as mediators and agents between various tissues. It is clear that the basic elements involved in polarization of macrophages (such as NO, ROS, sodium balance, and regulation of T-lymphocytes) have direct applicability and relevance to the development of essential hypertension. It is also readily apparent that the influence of the “neuro-immuno” axis on macrophages is profound and also versatile. Our evidence indicates a possible model for the role of macrophages in the development of hypertension, demonstrating a critical role for their ability to “switch” between phenotypes, almost as if on a “time-release program”.

Perhaps one of the most exciting and promising areas to be explored is the manipulation of macrophage polarization via the “neuro-immuno” axis. Although, results regarding renal denervation in patients are inconsistent (165, 166), a better understanding of the “neuro-immuno” axis may allow revisions to this or other therapies, including the development of novel pharmacological agents. In this pursuit, it will be extremely important to take into consideration the dynamic nature of macrophages and their ability to “switch” between phenotypes. This transition between phenotypes appears to be time-dependent, linked to the chronological course of the disease process. Thus, future studies should take into account the fact that although one phenotype, e.g. M1, dominates early in the course of the disease, another phenotype such as the M2 may dominate the later stages of a chronic disease process such as hypertension. Hence, the administration of therapies to patients targeting macrophage polarization will also be time-dependent. Although much remains to be discovered regarding the cholinergic, adrenergic, and neurohormonal receptor influence on macrophages in essential hypertension, novel therapeutic strategies that harness the power of macrophage polarization to reverse, control, or prevent essential hypertension and/or its devastating cardiovascular consequences have great promise.

Abbreviations

IFN-γ

interferon-gamma

IL-1b

interleukin-1 beta

IL-2

interleukin-2

IL-4

interleukin-4

IL-6

interleukin-6

IL-8

interleukin-8

IL-10

interleukin-10

IL-12

interleukin-12

IL-13

interleukin-13

IL-17

interleukin-17

IL-18

interleukin-18

IL-23

interleukin-23

TNF-α

tumor necrosis alpha

Ang II

angiotensin II

AT1R

angiotensin type 1 receptor

AT2R

angiotensin type 2 receptor

nAChR

nicotinic acetylcholine receptor

AR

adrenergic receptor

TLR

toll-like receptor

iNOS

inducible nitric oxide synthase

eNOS

endothelial nitric oxide synthase

nNOS

neuronal nitric oxide synthase

RAG

Recombination activating gene

DTR

Diphtheria toxin receptor

NO

nitric oxide

ROS

reactive oxygen species

TAM

tumor associated macrophages

MHC-II

major histocompatibility complex II

VEGF

vascular endothelial growth factor

TGF-β

tumor growth factor-beta

SHR

Spontaneously Hypertensive Rats

WKY

Wistar Kyoto

NK

Natural Killer Cell

LLT1

lectin like transcript 1

PPAR-γ

peroxisome proliferator-activated receptor-gamma

m-CSF

monocyte colony stimulating factor

PRR

pathogen-recognition receptor

PAMPs

pathogen-associated molecular patterns

DAMPs

danger associated molecular patterns

CNS

Central Nervous System

PNS

parasympathetic nervous system

SNS

sympathetic nervous system

a7-nAChR

alpha7-nicotinic acetylcholine receptor

klf4

kruppel-like factor 4

BBB

Blood-Brain Barrier

CP

Choroid Plexus

DC

dendritic cells

CVO

circumventricular organs

OVLT

organum vasculosum lamina terminalis

SFO

subfornical organs

AP

area postrema

PVN

paraventricular nucleus

RVLM

rostroventrolateral medulla

AV3V

anteroventral third ventricle

SCO

subcommissural organ

NTS

nucleus tractus solitarius

RAAS

renin-angiotensin-aldosterone system

MCP-1

monocyte chemoattractant protein-1

TONEBP

tonicity-enhancer binding protein

CRP

C-reactive protein

ICAM-1

intercellular adhesion molecule – 1

PBMC

peripheral blood monocytes

LPS

lipopolysaccharide

ACE

angiotensin converting enzyme