Paul J. Marvar

Inflammation, Immunity, and Hypertension

Aprominent pathology textbook used in the United States includes an image illustrating the renal histopathology caused by malignant hypertension. The legend describes striking “onion skin” changes of a renal arteriole. Curiously, a sea of mononuclear inflammatory cells surrounding this arteriole is overlooked both in the legend and in the related text. Moreover, nothing regarding inflammation or immune reactions is discussed. This lack of attention to inflammatory cells is, however, not surprising. Although many experimental studies have implicated inflammation in hypertension, these have largely been performed in experimental animals; there is no proof that inflammation contributes to human hypertension. In fact, some anti-inflammatory or immune-suppressing drugs (eg, nonsteroidal anti-inflammatory drugs and cyclosporine) paradoxically cause hypertension in humans, likely via off-target effects. Often the term “inflammation” is used in the context of cardiovascular disease as a catchall referring to nonspecific phenomena, such as elevation of C-reactive protein or the presence of macrophages in a tissue. Most clinicians and investigators find this vague and difficult to understand. Even more puzzling is that many studies now implicate the adaptive immune response, and in particular, lymphocytes, in hypertension and vascular disease. Traditionally, bacterial, viral, or tumor antigens activate this arm of immune defense. As such, it has been hard to imagine how adaptive immunity could be involved in a disease such as hypertension. In this article, we will attempt to address some of these puzzling questions. We will briefly review components of the innate and adaptive immune response, discuss data from many groups, including our own, that suggest that common forms of hypertension are immune mediated, and provide a working hypothesis of how signals from the central nervous system trigger an immune response that causes hypertension. ### General Concepts Regarding Inflammation and Immunity #### Innate Immunity The first line of defense against pathogens is the innate immune response. Important components of this system include epithelial …

Central and Peripheral Mechanisms of T-Lymphocyte Activation and Vascular Inflammation Produced by Angiotensin II–Induced Hypertension

Rationale: We have previously found that T lymphocytes are essential for development of angiotensin II–induced hypertension; however, the mechanisms responsible for T-cell activation in hypertension remain undefined. Objective: We sought to study the roles of the CNS and pressure elevation in T-cell activation and vascular inflammation caused by angiotensin II. Methods and Results: To prevent the central actions of angiotensin II, we created anteroventral third cerebral ventricle (AV3V) lesions in mice. The elevation in blood pressure in response to angiotensin II was virtually eliminated by AV3V lesions, as was activation of circulating T cells and the vascular infiltration of leukocytes. In contrast, AV3V lesioning did not prevent the hypertension and T-cell activation caused by the peripheral acting agonist norepinephrine. To determine whether T-cell activation and vascular inflammation are attributable to central influences or are mediated by blood pressure elevation, we administered hydralazine (250 mg/L) in the drinking water. Hydralazine prevented the hypertension and abrogated the increase in circulating activated T cells and vascular infiltration of leukocytes caused by angiotensin II. Conclusions: We conclude that the central and pressor effects of angiotensin II are critical for T-cell activation and development of vascular inflammation. These findings also support a feed-forward mechanism in which modest degrees of blood pressure elevation lead to T-cell activation, which in turn promotes inflammation and further raises blood pressure, leading to severe hypertension.

Induction of Hypertension and Peripheral Inflammation by Reduction of Extracellular Superoxide Dismutase in the Central Nervous System

The circumventricular organs (CVOs) lack a well-formed blood-brain barrier and produce superoxide in response to angiotensin II and other hypertensive stimuli. This increase in central superoxide has been implicated in the regulation of blood pressure. The extracellular superoxide dismutase (SOD3) is highly expressed in cells associated with CVOs and particularly with tanycytes lining this region. To understand the role of SOD3 in the CVOs in blood pressure regulation, we performed intracerebroventricular injection an adenovirus encoding Cre-recombinase (5×108 particles per milliliter) in mice with loxP sites flanking the SOD3 coding region (SOD3loxp/loxp mice). An adenovirus encoding red-fluorescent protein was injected as a control. Deletion of CVO SOD3 increased baseline blood pressure modestly and markedly augmented the hypertensive response to low-dose angiotensin II (140 ng/kg per day), whereas intracerebroventricular injection of adenovirus encoding red-fluorescent protein had minimal effects on these parameters. Adenovirus encoding Cre-recombinase–treated mice exhibited increased sympathetic modulation of heart rate and blood pressure variability, increased vascular superoxide production, and T-cell activation as characterized by increased circulating CD69+/CD3+ cells. Deletion of CVO SOD3 also markedly increased vascular T-cell and leukocyte infiltration caused by angiotensin II. We conclude that SOD3 in the CVO plays a critical role in the regulation of blood pressure, and its loss promotes T-cell activation and vascular inflammation, in part by modulating sympathetic outflow. These findings provide insight into how central signals produce vascular inflammation in response to hypertensive stimuli, such as angiotensin II.

The carotid body as a putative therapeutic target for the treatment of neurogenic hypertension

In the spontaneously hypertensive (SH) rat, hyperoxic inactivation of the carotid body (CB) produces a rapid and pronounced fall in both arterial pressure and renal sympathetic nerve activity (RSA). Here we show that CB de-afferentation through carotid sinus nerve denervation (CSD) reduces the overactive sympathetic activity in SH rats, providing an effective antihypertensive treatment. We demonstrate that CSD lowers RSA chronically and that this is accompanied by a depressor response in SH but not normotensive rats. The drop in blood pressure is not dependent on renal nerve integrity but mechanistically accompanied by a resetting of the RSA-baroreflex function curve, sensitization of the cardiac baroreflex, changes in renal excretory function and reduced T-lymphocyte infiltration. We further show that combined with renal denervation, CSD remains effective, producing a summative response indicative of an independent mechanism. Our findings indicate that CB de-afferentation is an effective means for robust and sustained sympathoinhibition, which could translate to patients with neurogenic hypertension.

Vascular Inflammatory Cells in Hypertension

Hypertension is a common disorder with uncertain etiology. In the last several years, it has become evident that components of both the innate and adaptive immune system play an essential role in hypertension. Macrophages and T cells accumulate in the perivascular fat, the heart and the kidney of hypertensive patients, and in animals with experimental hypertension. Various immunosuppressive agents lower blood pressure and prevent end-organ damage. Mice lacking lymphocytes are protected against hypertension, and adoptive transfer of T cells, but not B cells in the animals restores their blood pressure response to stimuli such as angiotensin II or high salt. Recent studies have shown that mice lacking macrophages have blunted hypertension in response to angiotensin II and that genetic deletion of macrophages markedly reduces experimental hypertension. Dendritic cells have also been implicated in this disease. Many hypertensive stimuli have triggering effects on the central nervous system and signals arising from the circumventricular organ seem to promote inflammation. Studies have suggested that central signals activate macrophages and T cells, which home to the kidney and vasculature and release cytokines, including IL-6 and IL-17, which in turn cause renal and vascular dysfunction and lead to blood pressure elevation. These recent discoveries provide a new understanding of hypertension and provide novel therapeutic opportunities for treatment of this serious disease.

Role of the NADPH Oxidases in the Subfornical Organ in Angiotensin II–Induced Hypertension

Reactive oxygen species and the NADPH oxidases contribute to hypertension via mechanisms that remain undefined. Reactive oxygen species produced in the central nervous system have been proposed to promote sympathetic outflow, inflammation, and hypertension, but the contribution of the NADPH oxidases to these processes in chronic hypertension is uncertain. We therefore sought to identify how NADPH oxidases in the subfornical organ (SFO) of the brain regulate blood pressure and vascular inflammation during sustained hypertension. We produced mice with loxP sites flanking the coding region of the NADPH oxidase docking subunit p22phox. SFO-targeted injections of an adenovirus encoding cre-recombinase markedly diminished p22phox, Nox2, and Nox4 mRNA in the SFO, as compared with a control adenovirus encoding red-fluorescent protein injection. Increased superoxide production in the SFO by chronic angiotensin II infusion (490 ng/kg min–1 ×2 weeks) was blunted in adenovirus encoding cre-recombinase–treated mice, as detected by dihydroethidium fluorescence. Deletion of p22phox in the SFO eliminated the hypertensive response observed at 2 weeks of angiotensin II infusion compared with control adenovirus encoding red-fluorescent protein-treated mice (mean arterial pressures=97±15 versus 154±6 mm Hg, respectively; P=0.0001). Angiotensin II infusion also promoted marked vascular inflammation, as characterized by accumulation of activated T-cells and other leukocytes, and this was prevented by deletion of the SFO p22phox. These experiments definitively identify the NADPH oxidases in the SFO as a critical determinant of the blood pressure and vascular inflammatory responses to chronic angiotensin II, and further support a role of reactive oxygen species in central nervous system signaling in hypertension.

Tyrosine metabolism during interferon-alpha administration: association with fatigue and CSF dopamine concentrations.

Chronic exposure to interferon (IFN)-alpha, an innate immune cytokine, produces high rates of behavioral disturbances, including depression and fatigue. These effects may be mediated by the actions of IFN-alpha on dopamine (DA) metabolism in the basal ganglia. Diminished conversion of phenylalanine (Phen) to tyrosine (Tyr), the primary amino acid precursor of DA, has been associated with inflammation, and may reflect decreased activity of the enzyme phenylalanine-hydroxylase (PAH). This study investigated the peripheral Phen/Tyr ratio in relation to cerebrospinal fluid (CSF) concentrations of DA and its metabolites in subjects treated with IFN-alpha plus ribavirin for hepatitis C and controls awaiting IFN-alpha therapy. Plasma Phen/Tyr ratios were significantly increased in IFN-alpha-treated subjects (n=25) compared to controls (n=9), and were negatively correlated with CSF DA (r=-0.59, df=15, p<0.05) and its metabolite, homovanillic acid (r=-0.67, df=15, p<0.01), and positively correlated with fatigue (r=0.44, df=23, p<.05) in IFN-alpha-treated patients but not controls. Given the role of tetrahydrobiopterin (BH4) in the PAH conversion of Phen to Tyr, CSF concentrations of BH4 and its inactive oxidized form, dihydrobiopterin (BH2), were examined along with CSF interleukin (IL)-6 in a subset of patients. BH2 concentrations were significantly increased in IFN-alpha-treated patients (n=12) compared to controls (n=7), and decreased CSF BH4 concentrations correlated with increased CSF IL-6 (r=-0.57, df=12, p<0.05). These results indicate that IFN-alpha is associated with decreased peripheral conversion of Phen to Tyr, which in turn is associated with reduced DA in the brain as well as fatigue. These alterations may be related to oxidation of BH4 secondary to IFN-alpha-induced activation of a CNS inflammatory response.

The Central Nervous System and Inflammation in Hypertension

In recent years a major research effort has focused on the role of inflammation, and in particular adaptive immunity, in the genesis of hypertension. Hypertension stimulates the accumulation of inflammatory cells including macrophages and T lymphocytes in peripheral tissues important in blood pressure control, such as the kidney and vasculature. Angiotensin II modulates blood pressure via actions on the central nervous system (CNS) and the adaptive immune system. Recent work suggests that the central actions of angiotensin II via the circumventricular organs lead to activation of circulating T-cells and vascular inflammation. The neuro-immune system plays an essential role in the pathogenesis of hypertension and further understanding of this relationship could lead to the development of new treatment strategies.

T lymphocytes and Vascular Inflammation Contribute to Stress-Dependent Hypertension

BACKGROUND Psychological stress is a significant risk factor for hypertension and also directly affects the immune system. We have previously reported that T lymphocytes are essential for development of hypertension and that the central nervous system contributes to peripheral T-lymphocyte activation and vascular inflammation in this disease; however, the role of T-cell activation in stress-related hypertension remains unclear. METHODS Wild-type and T-cell-deficient (RAG-1(-/-)) mice were subjected to daily episodes of stress and blood pressure was measured. Circulating T-cell activation markers and vascular infiltration of immune cells were analyzed, as were stress hormone levels and gene expression changes in the brain. The effects angiotensin II infusion in the presence of chronic stress was also studied. RESULTS Repeated daily stress contributed to acute elevations in blood pressure that were associated with increased activation of circulating T cells and increased vascular infiltration of T cells. Repeated stress increased blood pressure in wild-type but not RAG-1(-/-) mice. Adoptive transfer of T cells to RAG-1(-/-) mice restored blood pressure elevation in response to stress. Stress-related hypertension and vascular infiltration of T cells was markedly enhanced by angiotensin II. Moreover, angiotensin II-infused mice exposed to chronic stress exhibited greater blood pressure reactivity to an episode of acute stress. CONCLUSIONS These data demonstrate that stress-dependent hypertension triggers an inflammatory response that raises blood pressure at baseline and augments the hypertension caused by angiotensin II. These data provide insight as to how psychological stress contributes to hypertension.

Role of chemokine RANTES in the regulation of perivascular inflammation, T-cell accumulation, and vascular dysfunction in hypertension

Recent studies have emphasized the role of perivascular inflammation in cardiovascular disease. We studied mechanisms of perivascular leukocyte infiltration in angiotensin II (Ang II)‐induced hypertension and their links to vascular dysfunction. Chronic Ang II infusion in mice increased immune cell content of T cells (255 ± 130 to 1664 ± 349 cells/mg; P < 0.01), M1 and M2 macrophages, and dendritic cells in perivascular adipose tissue. In particular, the content of T lymphocytes bearing CC chemokine receptor (CCR) 1, CCR3, and CCR5 receptors for RANTES chemokine was increased by Ang II (CCR1, 15.6 ± 1.5% vs. 31 ± 5%; P < 0.01). Hypertension was associated with an increase in perivascular adipose tissue expression of the chemokine RANTES (relative quantification, 1.2 ± 0.2 vs. 3.5 ± 1.1; P< 0.05), which induced T‐cell chemotaxis and vascular accumulation of T cells expressing the chemokine receptors CCR1, CCR3, and CCR5. Mechanistically, RANTES–/– knockout protected against vascular leukocyte, and in particular T lymphocyte infiltration (26 ± 5% in wild type Ang II vs. 15 ± 4% in RANTES–/–), which was associated with protection from endothelial dysfunction induced by Ang II. This effect was linked with diminished infiltration of IFN‐γ‐producing CD8+ and double‐negative CD3+CD4–CD8– T cells in perivascular space and reduced vascular oxidative stress while FoxP3+ T‐regulatory cells were unaltered. IFN‐γ ex vivo caused significant endothelial dysfunction, which was reduced by superoxide anion scavenging. In a human cohort, a significant inverse correlation was observed between circulating RANTES levels as a biomarker and vascular function measured as flow‐mediated dilatation (R = –0.3, P < 0.01) or endothelial injury marker von Willebrand factor (R= +0.3; P< 0.01). Thus, chemokine RANTES is important in the regulation of vascular dysfunction through modulation of perivascular inflammation.—Mikolajczyk, T. P., Nosalski, R., Szczepaniak, P., Budzyn, K., Osmenda, G., Skiba, D., Sagan, A., Wu, J., Vinh, A., Marvar, P. J., Guzik, B., Podolec, J., Drummond, G., Lob, H. E., Harrison, D. G., Guzik, T. J. Role of chemokine RANTES in the regulation of perivascular inflammation, T‐cell accumulation, and vascular dysfunction in hypertension. FASEB J. 30, 1987–1999 (2016). www.fasebj.org