T Cell Immunometabolism and Redox Signaling in Hypertension

Abstract

Purpose of Review:

In this article, we summarize the current literature supporting metabolic and redox signaling pathways as important mechanisms underlying T cell activation in the context of hypertension.

Recent findings:

T cell immunometabolism undergoes dramatic remodeling in order to meet the demands of T cell activation, differentiation, and proliferation. Recent evidence demonstrates that the T cell oxidation-reduction (redox) system also undergoes significant changes upon activation, which can itself modulate metabolic processes and T cell function. Dysregulation of these signaling pathways can lead to aberrant T cell activation and inappropriate ROS production, both of which are linked to pathological conditions like hypertension.

Summary:

While the contribution of T cells to the progression of hypertension has been thoroughly investigated, how T cell metabolism and redox signaling changes, both separately and together, is an area of study that remains largely untouched. This review presents evidence from our own laboratory as well as others to highlight the importance of these two mechanisms in the study of hypertension.

Introduction

With the most recent update from the American College of Cardiology/American Heart Association now defining Stage 1 high blood pressure as having a systolic ≥130 mmHg over a diastolic ≥80 mmHg, the prevalence of hypertension among adults in the United States has jumped substantially to 46% [1]. Hypertension is the primary modifiable risk factor for the development of cardiovascular, renal, and cerebrovascular disease, making it the leading cause for mortality worldwide [2–4]. Despite advancements in research and available therapeutics, only 26% of hypertensive patients taking current standard-of-care medication achieve blood pressure control to recommended levels [5], increasing their risk of kidney, heart and vascular diseases [1, 6, 7]. Therefore, there is considerable need to understand the complex mechanisms underlying this significant human health crisis. While the study of the immunological component of hypertension, specifically T cells, has been a highly active area of investigation for the last 15 years, this review will focus exclusively on changes to immunometabolism and redox signaling as drivers of T cell function.

The History of T cells in Hypertension

The important contribution of T cells to the pathology of hypertension dates back to the 1970s, where thymus grafting into nude mice restored DOCA-salt-induced chronic hypertension and vascular disease similarly seen in wild-type mice [8]. This initial observation was followed by a 1980 study where normotensive recipient rats intravenously received splenic cells from hypertensive donors (either DOCA-salt or renal hypertensive) and developed elevated arterial hypertension compared to recipients receiving splenic cells from normotensive donors [9]. The field reemerged with the work by Rodríguez-Iturbe et al in 2002 where treatment with the immunosuppressant mycophenolate mofetil (MMF) normalized systemic hypertension in genetically hypertensive rats [10]. Interest in the role of T cells in hypertension was further rejuvenated in 2007 by Guzik et al, demonstrating that knockout of recombination-activating gene 1 (Rag1^−/−) and the lack of T and B cells protected mice from the full development of angiotensin II- and DOCA-salt-induced hypertension [11]. More importantly, adoptive transfer of T cells restored the development of hypertension in Rag1^−/− mice. This seminal paper ignited the entire field to explore the role of immunity and T cell signaling over the last 15 years [12–19].

Our laboratory was the first to elucidate the immunological component of salt-sensitive hypertension utilizing the Dahl Salt-Sensitive (SS) rat model. Salt-sensitive hypertension is a form of hypertension that is accompanied by particularly high morbidity and mortality [20, 21], and salt-sensitive hypertensive subjects have a 3-fold higher risk of developing cardiovascular diseases. The Dahl SS rat develops elevated blood pressure and renal end organ damage in response to a high salt diet [22–24], which importantly parallels the disease conditions observed in salt-sensitive hypertensive humans [25–29]. Treatment of SS rats with the immunosuppressive agent MMF reduced high salt-induced blood pressure and albuminuria, indicating the important role of the immune system in conferring salt-sensitivity [30]. It was observed that increasing dietary salt intake promoted T cell infiltration into the kidneys of SS rats, particularly around blood vessels in the renal cortex and vascular bundles in the renal medulla [31]. The lack of functional T and B cells via the genetic deletion of the Rag1 gene on the Dahl SS background resulted in blunted blood pressure, renal injury, and renal immune cell infiltration [32]. The specific contribution of T cells was demonstrated through deletion of the CD247 gene (encodes for the CD3 ζ chain of the T cell receptor) in the SS rat, which ablated CD3+ T cells, resulting in significantly reduced blood pressure and albuminuria in response to high salt challenge [33]. This provided evidence that functional T cells are specifically required for the full development of salt-sensitive hypertension. More recently, our laboratory has solidified the role of T cells in this salt-induced hypertensive response via adoptive transfer of wild-type SS splenocytes back into T cell-deficient SS^CD247−/− rats [34], where the specific reconstitution and enrichment of CD4+ T helper cells led to the exacerbation of salt-sensitive hypertension and renal damage compared to control SS^CD247−/− rats receiving PBS. This was the first demonstration of its kind in the Dahl SS rat.

Therefore, it is well-established that T cells are critical to the development of hypertension, but what remains largely unknown are the specific signaling mechanisms that occur within these infiltrating T cells that promote disease. Additional recent work from our laboratory indicates that T cell immunometabolic and redox signaling mechanisms may be critical for the full development of salt-sensitive hypertension [35, 36]. Metabolic pathways and redox reactions are at the core of all biological processes and the control of T cell activation and function via these two mechanisms will be the focus of this brief review.

Immunometabolism in T cells

Immunometabolism is defined as the intracellular metabolic pathways that determine the fate and function of immune cells. Technological advances in our ability to precisely define changes in these processes via highly sensitive transcriptomic and metabolomic approaches have reinvigorated the study of metabolism by immunologists, giving rise to this field over the last decade. The study of immunometabolism specifically in T cells has been wonderfully and extensively reviewed [37, 38]. T cells require highly adaptive metabolic processes in order to maintain the continuous cycling between quiescent and activated states. Depending on the state, T cells rely primarily on two main metabolic processes to generate energy: oxidative phosphorylation (OXPHOS) and glycolysis. In the quiescent state, mature naïve T cells utilize OXPHOS for the generation of ATP [39] and critically depend upon the cytokine IL-7 for glucose uptake and the homeostatic proliferation, maintenance, and survival of naïve T cells [40]. Upon activation via T cell receptor ligation and costimulation, T cells undergo significant metabolic reprogramming characterized by aerobic glycolysis to support anabolic processes. Despite the oxygen-rich environment and less efficient ATP production compared to OXPHOS, aerobic glycolysis importantly produces critical intermediates required for nucleotide, amino acid, and reducing metabolite biosynthesis [41]. This T cell metabolic reprogramming and energy switch is central to the biosynthesis, cell growth, differentiation, and proliferation necessary to carry out its effector functions. Once the stimulus is removed and pathogen cleared, a small subset of these activated T cells will survive and shift back to a quiescent memory state, once again relying heavily on OXPHOS metabolism. However, memory T cells are metabolically reprogrammed with greater mitochondrial mass and substantially more spare respiratory capacity compared to naïve T cells to allow for their rapid recall and response to a reintroduced pathogen [42].

Exploration down to this level of T cell metabolism and activation in the context of disease is largely lacking in the field of hypertension. We have systematically investigated, at the transcriptomic level, how the T cell specifically changes in response to diet, salt, and location within a whole animal model of salt-sensitive hypertension. SS rats maintained on a whole grain, plant-based diet were considerably protected from the development of hypertension and renal damage compared to SS rats maintained on a casein, animal protein-based diet, despite being challenged to same high salt diet [43]. This interestingly corresponded with lesser immune cell infiltration into the kidneys of the rats fed the grain-based diet, and given our specific interest in T cells [31–33], we sought to transcriptionally phenotype T cells from both the periphery and kidney utilizing an RNAseq approach [35]. Our first major observation was the clear and remarkable activation of genes when comparing T cells from the periphery versus those infiltrating the kidney. Independent of diet or salt, over 50% of all annotated genes were found to be significantly differentially expressed between blood and kidney, indicative of a metabolic switch or ‘turning on’ from a quiescent to activated state upon recruitment to the site of injury. Second, the renal T cells isolated from the disease-prone SS rats fed the casein, animal-based diet were characterized by changes in genes associated with a distinctive proinflammatory phenotype (Figure 1). Ingenuity and KEGG Pathway Analysis revealed an overrepresentation of canonical pathways specifically related to T cell receptor signaling, T helper cell signaling, and the Th2 pathway. While this would seem expected, when looking at the significant pathways in the protected grain-fed SS rats, we did not simply observe an opposing downregulation of inflammatory genes. Instead, renal T cells isolated from the protected SS rats fed the grain, plant-based diet revealed a metabolically suppressed profile in response to high salt, with significant downregulation of pathways related to nucleotide, amino acid, and protein metabolism (Figure 1). While these results are of course only reflective of T cell metabolism at the transcript level, it is clear that gene activation in T cells is separate and distinct between these two hypertensive phenotypes, and whether these changes are cause or consequence of the disease remains to be elucidated. Determination of how these alterations to the T cell transcriptome translate to changes in metabolites, metabolic activity, and ultimately effector function of T cells is a current area of active investigation in our laboratory.

Figure 1.

Kidney T cell transcriptome analysis of differentially expressed genes via Ingenuity Pathway Analysis (IPA) or Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed enrichment of pro-inflammatory pathways related to T cell signaling in disease-prone SS rats fed an animal-based diet. Conversely, kidney T cell derived from disease-protected SS rats fed a plant-based diet revealed enrichment of gene pathways primarily related to metabolic processes. Figure redrawn with permission from Abais-Battad et al [35].

Redox Signaling in T cells

The generation of reactive oxygen species (ROS) in cells were once considered to be a purely detrimental occurrence, since free radicals are known to induce irreversible damage to DNA, proteins, and lipids. However, it is now widely accepted and demonstrated that controlled levels of ROS have very specific roles in normal cellular signaling, whereas uncontrolled ROS production or ‘oxidative stress’ is correlated with a number of pathological conditions, including hypertension [44]. While studies examining the contribution of endogenously produced ROS in T cells are relatively new [45], its homeostatic role in T cell activation, metabolism, and function is clear. In addition to ROS being unavoidably generated from OXPHOS, another major source of ROS in T cells comes from NADPH oxidase (NOX), with T cells being shown to express NOX2 [46, 47]. Jackson et al provided early evidence that phagocytic NOX2 is activated upon T cell receptor (TCR) stimulation, and the generation of this appropriate, low-level ROS in T cells from both NOX activity as well as mitochondrial OXPHOS enhances, sustains, and regulates TCR signaling [47, 48] to promote the metabolic rewiring necessary for the controlled switch between the quiescent and effector states. If left unchecked by antioxidant systems, continued and excessive ROS production results in defective metabolic rewiring, aberrant T cell function and response [49, 50], and ultimately pathological conditions. While studies utilizing a systemic knock-out approach of various NOX2 subunits (p47^phox, gp91^phox) certainly demonstrated that NADPH oxidase-derived ROS is critical for T cell polarization and skewing the Th response [47, 51], a major issue is the inability to dissect whether these results are direct effects of NOX2 loss within T cells or are global, non-T cell effects due to loss of NOX2 throughout the entire host.

It is well-documented that the inappropriate production of ROS is associated with the development of hypertension and cardiovascular disease [44, 52], and in the Dahl SS rat, increased production of superoxide via NADPH oxidase in the renal medulla is linked to the progression of salt-sensitive hypertension and renal damage [53, 54]. While the null mutation of the p67^phox gene on the Dahl SS background (SS^{p67phox−/−}) was the first evidence that ROS specifically derived from the NOX2 isoform of NADPH oxidase is critical for the development of salt-sensitive hypertension [55], these studies are again limited by the use of a systemic p67^phox knockout. With the infiltration of immune cells into target organs considered to be central to the development of hypertension [22] and with NOX2 known to be highly expressed on immune cells [56], it is conceivable that these immune cells serve as a critical source of ROS contributing to disease progression. Therefore, our laboratory first began the investigation of NOX2 in immune cells by determining whether infiltrating T cells isolated from the SS rat kidneys express NOX2 subunits. Indeed, these renal T cells highly express p67^phox, gp91^phox, p22^phox, and p47^phox, especially upon exposure to high salt, indicating the presence of the molecular machinery necessary for NOX2-derived ROS production [46]. To importantly dissociate the contribution of ROS from immune cells versus the renal parenchyma, we utilized a total body irradiation/bone marrow transfer approach to transfer functional, ROS-producing SS wild-type bone marrow cells into irradiated SS^{p67phox−/−} recipients to generate a chimeric rat with the ability to produce NOX2-derived ROS only in cells of the hematopoietic lineage [36]. This technique addresses the major issue of dissecting the effect of global versus immune cell NOX2 function. We discovered that the production of NOX2-derived ROS solely in cells of hematopoietic origin (immune cells), but absent in the remainder of the host, was sufficient to drive and exacerbate hypertension and renal damage, highlighting the fundamental role of immune cell ROS in the pathogenesis of salt-sensitivity (Figure 2). While the further determination of the specific immune cell type remains the current focus of our investigation, these results demonstrate the importance of the exact cellular localization and source of ROS production, which becomes critical when developing effective antioxidant therapies.

Figure 2.

Global genetic deletion of the p67^phox subunit of NOX2 attenuates salt-induced hypertension and renal damage in SS^{p67phox−/−} rats (A). To delineate the contribution of free radical production from immune cells versus that of parenchymal cells, we generated a chimeric rat where SS^{p67phox−/−} rats received SS bone marrow and p67^phox would only be expressed in cells of hematopoietic origin (immune cells). Shown in (B), intact p67^phox and functional NOX2 in immune cells was sufficient to restore hypertension and renal damage to SS^{p67phox−/−} rats lacking p67^phox in the parenchyma. *p<0.05, **p<0.005, ***p<0.001 SS^{p67phox−/−} vs SS. Data redrawn with permission from Abais-Battad et al [36]. Reprinted from Free Radical Biology and Medicine, Vol. 146, Justine M. Abais-Battad, Hayley Lund, John Henry Dasinger, Daniel J. Fehrenbach, Allen W. Cowley, and David L. Mattson, NOX2-derived reactive oxygen species in immune cells exacerbates salt-sensitive hypertension, 333-339, 2020, with permission from Elsevier

Therapeutic Implications of Immunometabolic Modulators and Antioxidants

The extensive beneficial effects of a plant-based diet to human health and disease are recognized not only within the scientific community but the general public as well. What is less recognized are the specific plant components and underlying mechanisms driving this protection in humans. A few clinical studies have attempted to address the effect of plant-based or Mediterranean diets on immunity and T cell function, but have provided contradictory results. Adults at high risk of cardiovascular disease saw a significant anti-inflammatory effect with a 5-year Mediterranean diet intervention, to include a reduction in adhesion molecule expression on circulating T cells [27440261]. In other studies, a vegetarian or vegan diet had no effect on number or function of circulating T cells, specifically [2072829, 32147197]. One major limitation among these human studies is the use of circulating peripheral blood mononuclear cells to assess T cell function; these cells may not accurately reflect the inflammatory milieu that exists in target organs, which may account for the discrepancy in results between these studies. As demonstrated in our work, circulating T cells display a drastically different transcriptional profile compared to T cells found in the kidney, highlighting the importance of cellular localization when performing these analyses on immune cell function and metabolism.

In terms of targeting redox signaling, the use of antioxidants for treatment have held great promise in numerous pre-clinical animal models; however, there have been underwhelming effects of antioxidants in human trials [22419320, 18474268] due to short trial duration and poor antioxidant bioavailability [19694341, 30121293]. The studies discussed in this review again demonstrate the importance of cellular localization as well as source of ROS production, which becomes critical when determining targets for potential therapeutics. In light of this work, perhaps taking a more targeted approach to deliver high enough concentrations of antioxidants to the cells specifically responsible for producing deleterious ROS would lead to more efficacious therapies and ultimately better outcomes in humans. While no such therapies currently exist to specifically target T cell metabolism or ROS production, this is a future area of drug development with great potential.

Conclusions

In this review, we have briefly summarized the recent evidence regarding the role of metabolic and redox signaling in the determination of T cell function, and its implications for pathological conditions like hypertension, specifically salt-sensitivity. However, the reality is that immunometabolism and redox mechanisms are highly interdependent and tightly interwoven with one another, where metabolic reprogramming can alter ROS and vice versa, ROS can affect immune cell function via direct effects on metabolic enzymes and metabolites. In order to understand the complexities that occur during pathological conditions, thorough investigation of these redox-metabolism interactions in the regulation of immune function remains a critical area of future research.