Recent Advances on Immunity and Hypertension: The New Cells on the Kidney Block

Abstract

Over the last 50 years, contribution of the immune system has been identified in the development of hypertension and renal injury. Both human and experimental animal models of hypertension have demonstrated that innate and adaptive immune cells, along with their cytokines and chemokines, modulate blood pressure fluctuations and end organ renal damage. Numerous cell types of the innate immune system, specifically monocytes, macrophages, and dendritic cells present antigenic peptides to T cells promoting inflammation and the elevation of blood pressure. These T cells and other adaptive immune cells, migrate to vascular and tubular cells of the kidney and promote end-organ fibrosis, damage, and ultimately hypertensive injury. Through the development of high throughput screening, novel renal and immune cell subsets have been identified as possible contributors and regulators of renal injury and hypertension. In this review, we will consider classical immunological cells and their contribution to renal inflammation, and novel cell subsets, including renal stromal cells, that could potentially shed new light on renal injury and hypertension. Lastly, we will discuss how interorgan inflammation contributes to the development of hypertension and hypertension-related multi-organ damage, and the clinical implications of the immunological components of renal injury and hypertension.

Graphical Abstract

INTRODUCTION

Hypertension is a major contributor for the development of cardiovascular morbidity and mortality and is strongly linked to the development of renal injury (1, 2). Importantly, approximately 50% of people worldwide have hypertension, with an excess of ~23 million patients developing chronic kidney disease (1, 2). Over the last 50 years, it has been established that inflammation and immune cells contribute to the development of hypertension and subsequent renal injury (3–5). Numerous studies have demonstrated in experimental models of hypertension that modulating or inhibiting the immune system prevents elevation in blood pressure and renal injury (3, 4, 6). While it is well-demonstrated that classical physiological mechanisms contribute to the development of hypertension, including endothelial cell dysfunction (7), impaired vascular function (8), and dysregulation of the sympathetic nervous system (9), these multiple mechanisms fall outside the scope of this review. However, the immune system and inflammatory signaling mechanisms of hypertension affect multiple organ systems, specifically the kidney, and thus the development of subsequent renal injury. It is acknowledged that inflammation is complex, vast, and dichotomous; however, this review will take a closer look at the pivotal role of classical and novel immune cell subtypes, multi-organ inflammatory crosstalk, and co-morbidities play in the inception and development of hypertension-induced renal injury.

CLASSICAL IMMUNE CELLS IN HYPERTENSION AND RENAL DYSFUNCTION

Immunology and the immune cell response consists of two broad categories, referred to as innate and adaptive immunity (10, 11). The innate immune system is the first line of defense against pathogens and plays a critical role in controlling infections and maintaining homeostasis. The main cell types of the innate immune system consist of antigen presenting cells (APCs) such as macrophages, dendritic cells (DCs), neutrophils, and natural killer (NK) cells (12). Regarding hypertension, these innate cells have been widely explored in terms of their dysregulation during pathological states which has been proposed to lead to chronic vascular inflammation and subsequent dysfunction (13). However, the innate immune system cannot facilitate the development of hypertension alone, and the adaptive immune system, T and B cells, also play a critical role in hypertension (14, 15). Herein, we explore the classical immunological role that each of these cell types contribute to hypertension development and renal injury (Figure 1).

Figure 1: Classical Immunology in Development of Hypertension and Renal Inflammation.

Innate and adaptive immune cells have been implicated in the generation and development of renal inflammation and hypertension. Over the past two decades, antigen presenting cells (APCs), T cells, and B cells have been well characterized for their role in pathogenesis of renal inflammation and high blood pressure.

Innate Immunity in Hypertension and Renal Inflammation

Monocytes/Macrophages:

Macrophages are essential to the function of the innate immune system and are found throughout the body and vascular tissues. They work by engulfing pathogens and cellular debris such as damage-associated molecular patterns (DAMPs) via phagocytosis during infections and cellular injury (16, 17). When encountering pathogens or DAMPs, macrophages can class switch from M2 (anti-inflammatory) to M1 (pro-inflammatory) (18). Using pharmacological immunosuppression with mycophenolate mofetil (MMF) during angiotensin II (Ang II) infusion + high salt diet (4% NaCl) of Sprague-Dawley rats, Franco et al. found a profound reduction in systolic blood pressure and renal interstitial lymphocytes and macrophages(19). In subsequent studies, inhibition of chemokine receptor 2b (CCR2b), which is expressed on macrophages, resulted in a reduction in renal macrophages in rats treated with Ang II + high salt diet (20), highlighting the effects of pharmacological blockade of macrophages on hypertensive renal injury. In a seminal study by Wenzel et al., it was demonstrated that selective deletion of lysozyme M-positive monocytes/macrophages prevents immune cell infiltration, hypertension and subsequent vascular dysfunction (21). Moreover, utilizing a functionally deficient macrophage murine model, osteoporotic (Op/Op) mice, numerous studies indicated a reduction in hypertension in response to both Ang II and deoxycorticosterone acetate (DOCA) salt (22, 23). In hypertensive Dahl salt-sensitive (Dahl SS) rats, it has been demonstrated that salt-sensitive hypertension promotes an M1-like phenotype with accumulation around injured glomeruli and medullary tubules (24). In a recent translational study, Loperena et al. demonstrated that hypertensive stretch of human aortic endothelial cells (10%) induced interleukin-6 (IL-6) secretion promoting the transformation of classical monocytes (CD14⁺⁺/CD16⁻) to activated pro-inflammatory intermediate monocytes (CD14⁺⁺/CD16⁺) (25). These activated intermediate monocytes secreted large amounts of pro-inflammatory cytokines like IL-6, tumor necrosis factor alpha (TNF-α), interleukin-23 (IL-23) and interleukin-1 beta (IL-1β) (25). In accordance with these studies, the importance of monocytes/macrophages has been clinically demonstrated, where hypertensive human subjects have increased circulating monocytes/macrophages compared to healthy controls. Moreover, these studies demonstrated an increase in pro-inflammatory cytokines like TNF-α and IL-6, which positively correlated with blood pressure (25–32). Taken together, these basic, translational, and clinical studies demonstrate an important role of monocytes/macrophages in the development of hypertension and renal inflammation.

Dendritic Cells:

DCs serve as the main professional antigen presenting cell, responsible for bridging the innate and adaptive immune systems. This bridging is performed through antigen presentation with major histocompatibility complex II (MHC II) to adaptive immune cells such as T cells (33, 34). Over the last 10 years, it has been demonstrated that DCs play a critical role in the development of hypertension (35–37). Vinh and colleagues demonstrated that inhibition of co-stimulatory proteins, specifically cluster of differentiation 80 (CD80) and cluster of differentiation 86 (CD86) with cytotoxic T-lymphocyte associated protein 4 (CTLA4)-IgG, prevented both Ang II-induced and DOCA-salt hypertension (38). Moreover, the authors demonstrated that intervention with CTLA-IgG reserved the elevations in systolic blood pressure in response to DOCA-salt hypertension, demonstrating a key role of antigen presentation in the development of hypertension and renal injury (38). In a seminal study by Kirabo et. al, it was shown that the highly reactive lipid peroxidation products, isolevuglandins (IsoLGs), accumulate in DCs and promote renal inflammation and fibrosis (35). Moreover, treatment with the IsoLG scavenger, 2-hydroxybenzilamine (2-HOBA), prevented the accumulation of IsoLGs in DCs and subsequent renal fibrosis and inflammation (35). Moreover, numerous studies have demonstrated a profound increase in DC accumulation in the kidneys in both Ang II-induced and salt-sensitive hypertension (36, 37, 39, 40). Crowley and colleagues demonstrated that Fms-like tyrosine kinase 3 ligand (FLT3L), a critical component of classical DC differentiation, is necessary for the development of Ang II-induced hypertension through renal oxidative stress and renal T cell accumulation (39). Van Beusecum et al. demonstrated that DC-specific knockout of the salt-sensing kinase, serum-glucocorticoid kinase 1 (SGK1) prevented the development of salt-sensitive hypertension, renal fibrosis, and renal immune cell infiltration in compared to controls (36). In accordance with salt-sensitive hypertension, Barbaro et al. demonstrated a critical role of epithelial sodium channel (ENaC) on DC activation and the promotion of blood pressure in response to low dose Ang II (37). Importantly, studies have investigated the role of suppressing DC activation and subsequent T cell activation. Lu et al. demonstrated that the ubiquitin-editing protein A20, is critical in suppressing the activation of DCs and subsequent T cell activation in the development of Ang II-induced hypertension (40). Furthermore, they demonstrated that A20 knockout increased renal accumulation of memory T cells, suggesting that A20 suppresses the activation of DCs and renal inflammation in hypertension (40). These studies and others have demonstrated a critical role of DC activation, subsequent T cell activation in hypertension-induced renal inflammation and damage.

The development of single cell ribonucleic acid (scRNA)-sequencing, spatial transcriptomics, and proteomics, has made it abundantly clear the innate immune system is a fluid system with numerous immune cell subsets having overlap of “defining” surface expression markers. Specific cell-defining populations as M1-like, M2-like, classical DCs, classical, intermediate, and non-classical monocytes, may need to be revisited and redefined by their surface expression markers and cytokines produced into the broader category of APCs. Further research and agreement within the scientific community is required to provide standardized nomenclature for these complex cells.

Adaptive Immunity in Hypertension and Renal Inflammation

T Cells:

T cells are classically identified as surface expression of cluster of differentiation 45 (CD45) and the T cell receptor (TCR), cluster of differentiation (CD3), and they are divided into two classical subpopulations, T Helper cells (Th) with expression of cluster of differentiation 4 (CD4⁺) and cytotoxic T cells with expression of cluster of differentiation 8 (CD8⁺). Both cell types are involved in the maturation and response of the adaptive immune system. The importance of T cells in the development of hypertension was first established in 1977, utilizing athymic mice, which resulted in the prevention of hypertension-induced injury (41). Moreover, numerous groups demonstrated that immunosuppression of CD3 with mycophenolate mofetil (MMF), prevented renal T cell infiltration and accumulation in multiple murine models of hypertension (42–47). Mattson and colleagues have demonstrated that treatment with MMF prevents salt-sensitive hypertension in Dahl SS rats through a reduction of renal T cell infiltration (44, 48). Recently, Maranon et al. demonstrated that MMF treatment and immunosuppression of T cells have sex-dependent effects in spontaneously hypertensive rats (SHRs), highlighting key studies on the role of T cells in rats in the development of hypertension (47). In a seminal study by Guzik et al., they demonstrated that deletion of both T and B cells using the recombination-activating gene1 knockout (Rag1^−/−) mouse, prevented the elevation in blood pressure in response to Ang II infusion (49). When CD3⁺ T cells were adoptively transferred into Rag1^−/− mice, the blood pressure phenotype from Ang II infusion was restored, highlighting a critical role of CD3⁺ T cells in the development of hypertension (49). Additionally, Crowley et al. demonstrated that lack of lymphocytes (CD3⁺ cells), prevented renal inflammation, fibrosis, and the elevation of blood pressure through renal sodium excretion (50). Building upon these studies, numerous groups have demonstrated key sex differences in the role of CD3⁺ T cells on renal inflammation and the development of hypertension (51–53). Adoptive transfer of male CD3⁺ T cells into Rag1^−/− mice led to increased renal immune cell infiltration compared to female CD3⁺ T cells, demonstrating a key sex difference (51–53). These studies laid the foundation for the role of CD3⁺ T cells in the development of renal inflammation and hypertension and paved the way for numerous groups to investigate the contribution of both CD4⁺ and CD8⁺ T cells in the vascular endothelium and in the kidney (54–56).

CD8⁺ T cells are also known as cytotoxic T cells for their ability to directly kill infected and/or damaged cells via major histocompatibility complex I (MHC I)-mediated activation. This mechanism is critical in maintaining homeostasis via the clearance of dysfunctional cell types that may promote pathological states in the body. Importantly, numerous studies have identified a critical role for CD8⁺ T cells in the development of hypertension and renal injury (57–60). Trott et al. demonstrated that CD8^−/− mice were protected from renal vascular remodeling and prevented sodium and water retention during Ang II infusion. Moreover, the adoptive transfer of CD8⁺ T cells to Rag1^−/− conferred hypertensive renal injury and blood pressure elevations in response to Ang II infusion (57). In accordance with these studies, it has been demonstrated that pro-inflammatory CD8⁺ T cells are elevated in patients with hypertension compared to normotensive controls (58). Lastly, in a murine model of salt-sensitive hypertension, DOCA-salt hypertension, it was demonstrated that CD8⁺ T cells directly influence the upregulation of Na-Cl cotransporter (NCC) in the distal convoluted tubule, highlighting a critical importance of CD8⁺ T cells on sodium reabsorption in salt-sensitive hypertension (60).

CD4⁺ T cells are also known as T_H cells due to their supportive function in coordinating the immune system’s response to activation mainly through the two subsets of T_H cells, specifically T_H1 and T_H17 subtypes. These subtypes secrete several pro-inflammatory cytokines (interferon-gamma (IFN-γ), interleukin-17 (IL-17), and TNF-α) that can activate cell types in both the innate and adaptive immune system thereby increasing the inflammatory response to hypertension. CD4⁺ T cells are hypothesized to be crucial for the maintenance of chronic hypertensive states. In the key study by Trott et al., the authors demonstrated that adoptive transfer of CD4⁺ T cells to Rag1^−/− did not confer elevations of blood pressure, renal inflammation or end organ damage, though key studies have demonstrated that CD4⁺ T cells are increased in the kidneys of hypertensive male mice in both Ang II infusion and salt-sensitive models (61–63). Activated CD4⁺ T cells secrete the pro-inflammatory cytokine IL-17, promoting sodium reabsorption and renal injury in the development of hypertension (62, 63). Norlander et al. demonstrated that loss of the salt-sensing kinase SGK1 in T cells prevented renal inflammation and end-organ damage in both Ang II-induced and DOCA-salt hypertension (62). These studies taken together demonstrate a pro-hypertensive role for CD4+ T cells.

Hypertension and renal inflammation are not only increased in pro-inflammatory immune cells, but it can also be the result of a reduction in anti-inflammatory immune cells, including T regulatory cells (T_regs). T_regs are CD4⁺ T cells that have surface expression of CD25 and intracellular expression of forkhead box P3 (Foxp3), and they are seen as the “brakes” to inflammatory processes. It has been well-documented that T_regs are elevated in females compared to males, and they play a protective role in salt-sensitive and essential hypertensive murine models (64–66). Not only are T_regs elevated in females compared to males, but knockdown of Foxp3⁺ T_regs eliminate the protective effects seen in wildtype female mice (67), indicating a reduction in T_regs could promote the development of hypertension and renal inflammation.

In addition to the canonical mechanisms by which the immune system contributes to hypertension and end organ damage, there has been more recent explorations into extra-renal mechanisms that contribute to hypertension that involve the immune system. Over the last 10 years, it has been recognized that hypertension leads to an increase in renal sodium (Na⁺) reabsorption, and that this Na⁺ retention does not simply lead to water retention (68). Utilizing a Na+-MRI to detect relative amounts of interstitial tissue compartments, numerous studies have demonstrated that Na⁺ is retained and accumulates in the skin, muscle, and brains of both animals and human subjects (69–72). In accordance with these studies, it has been demonstrated high Na⁺ concentrations can promote T cell differentiation of CD4⁺ T cells to T_H17 cells and promote IL-17 and IFN-y production (62, 73–75). Specifically, Wu et al. and Norlander et al. found that high Na⁺ concentrations promote the differentiation of naïve CD4⁺ T cells into pathogenic T_H17 cells through an SGK1-mediated signaling pathway (62, 73). Moreover, Wilck et al. demonstrated in a murine model of high Na⁺ diet feeding and human subjects fed a high Na⁺ diet, that high Na⁺ reduces Lactobacillus in the gut microbiome, which in turn promotes the differentiation to pathogenic T_H17 cells (75). Furthermore, treatment of either mice or human subjects with Lactobacillus-rich probiotics prevented the elevations in blood pressure and the T cell polarization to a T_H17 cell phenotype. These data provide critical evidence of the role of elevated Na⁺ in the development of hypertension, and link the importance of interorgan communication, specifically the gut/immune/kidney axis in the development of hypertension and renal injury, which will be further discussed in upcoming sections.

B Cells:

The role of B cells in the development of renal inflammation and hypertension remains to be fully elucidated. While clinical studies have suggested a role of B cell antibodies in the development of blood pressure elevations, Guzik et al. demonstrated that adoptive transfer of B cells alone into Rag1^−/− recipient mice did not affect the development of Ang II-induced hypertension, therefore suggesting B cells may have little impact in the development of essential hypertension (49). However, in an elegant study by Chan et al., they found that B cell depletion with anti-cluster of differentiation 20 (CD20) antibody blunted the response to Ang II-induced hypertension by approximately 30% (76). Using a genetic B cell depletion approach, B-cell-activating factor receptor (BAFFR) knockout mice, BAFFR^−/−, conferred a dampened systolic blood pressure elevation response to Ang II, similar to anti-CD20 antibody treatment (76). However, Chen et al. found in contrast that mice with severe global deficiency in B cells (μMT^−/− mice), were not protected from elevations in blood pressure from Ang II infusion or DOCA-salt sensitive hypertension (77). Moreover, μMT^−/− mice exhibited no protection from renal inflammation and damage (77). It is highly important to recognize that there is T and B cell crosstalk in the pathogenesis of hypertension and renal injury. Taylor et al. demonstrated that depletion of plasma cells with bortezimib in New Zealand Black White F1 (NZBWF1) mice, reduced blood pressure and prevented renal immune cell infiltration in a murine model of lupus-induced hypertension (Taylor et al. 2018 Hypertension), suggesting a critical role of B cells in activating the T cell-dependent pathogenesis of hypertension.

NEW IMMUNE CELLS ON THE BLOCK

The seminal work from Guzik et al highlighted the role of T cells in hypertension and cardiovascular disease, which led to a rise in the number of studies linking the contribution of immunity to the development of hypertension (49). Since this time, numerous cell types have been identified and explored as pro- or anti-hypertensive, which has been thoroughly described in other reviews (15, 78). With the help of new technologies such as transcriptomics, single cell ribonucleic acid sequencing (RNAseq), and mass cytometry by time of flight (CyTOF), new cell types have been identified. However, an important consideration is that the current nomenclature of immune cell subsets may be a limiting factor in identifying novel immune populations involved in the pathogenesis of hypertension. As our investigatory tools become more refined and specific, this could potentially help separate out subpopulations of immune cells through a different nomenclature than just cell surface markers. While this list is not inclusive of all cell types, this section will highlight some of the more recently investigated cell types that contribute to hypertension and cardiovascular disease, and how they might work in concert with other immune cells (Figure 2).

Figure 2: Novel Renal and Immune Cells in the Development of Renal Inflammation and Hypertension.

(A) Novel innate immune cells have been demonstrated to orchestrate inflammatory responses in both renal inflammation and hypertension including kidney stromal antigen presenting cells (APCs), specifically podocytes, endothelial cells, tubular epithelial cells (TECs), and novel myeloid cells including DC subsets and neutrophils. (B) Novel adaptive immune cell types including natural killer T cells (NKT cells), and T cells have been recently identified to play a role in the regulation of blood pressure and renal inflammation.

Novel T Cell Populations:

Gamma Delta (γδ) T cells are considered an innate lymphoid cell type which express γδ TCR chains instead of conventional Alpha Beta (αβ) TCR chains (79). Unique in the fact that they can act as effector cells and innate-like cells, γδ T cells function as antigen presenting cells similarly to DCs and produce IL-17 and IFN-γ (80). In recent years, there is data demonstrating how γδ T cells contribute to hypertension and end-organ damage in pre-clinical models (63, 81, 82). In a mouse model of preeclampsia (PE), genetic deletion of γδ T cells protects these mice from PE-like symptoms, suggesting these cells could contribute to pregnancy-induced hypertension (83). Furthermore, Chatterjee et al demonstrated that γδ T cells are significantly elevated in placentas from women that experienced a preeclamptic pregnancy versus a normotensive pregnancy. There is even clinical data supporting a role for γδ T cells in human hypertension. In the Multi-ethnic Study of Atherosclerosis (MESA), there was an association with average systolic blood pressure and the number of circulating γδ T cells (11). A recent study by Berillo et al further utilized singe cell RNA-sequencing to identify subsets of γδ T cells in both Ang II-induced hypertensive mice and hypertensive human subjects (84). Comeau et al demonstrated that memory γδ T cells develop in response to a hypertensive stimulus in target tissues such as blood vessels, perivascular adipose tissues, and lymph nodes (85). This was discovered by exposing male C57BL/6J mice to a model of pressor and subpressor dose Ang II for two weeks each before isolating memory γδ, CD4+ and CD8+ T cells. Adoptive transfer of memory γδ T cells versus sham γδ T cells elicited a greater hypertensive response due to memory γδ T cells. This field is continuing to evolve through the discovery of γδ T cell subsets and their role in Ang II-induced hypertension. By treating Ang II-infused C57BL/6J mice with various monoclonal antibodies targeting the subsets of γδ cells, Mahmoud et al demonstrated that Vγ6/ Vδ1+ γδ T cells reduce blood pressure and improve endothelial dysfunction in these mice (86). Deeper investigations are certainly needed to fully understand the contributions of these subsets in the development of hypertension.

Natural killer T cells (NKT) are a special subset of γδ T cells that co-express NK and T cell receptors. These cells are considered innate-like T cells that can be activated via two pathways: 1) direct activation through cluster of differentiation 1d (CD1d)-dependent mechanisms and 2) indirect activation through cytokine stimulation from toll-like receptor (TLR)-activated APCs. In terms of hypertension, deletion of CD1d in male C57BL/6 mice resulted in exacerbated blood pressure in two models of hypertension, Ang II and deoxycorticosterone acetate (DOCA)-salt induced (87). Furthermore, a bone marrow transplant (BMT) experiment demonstrated that cells of hematopoietic origin elicited these pro-hypertensive responses. Recipient mice were irradiated with a lethal dose of 8.5Gy to ablate the bone marrow before being injected with either C57BL/6 or CD1dko bone marrow (1x10⁷ cells). Adoptive transfer of hematopoietic from the CD1dko donors resulted in an exacerbation of blood pressure response to Ang II infusion relative to mice receiving bone marrow from C57BL/6 donors. Similarly to the mouse investigations of NKT cells, Xiao et al demonstrated that hypertensive humans exhibit a reduction in number of NKT cells relative to normotensive subjects. If NKT cells were activated with alpha-galactosylceramide (a specific activator of NKT cells), there was an attenuation in Ang II-induced hypertension and associated end-organ damage (88).

Follicular helper T cells (TfH) are identified as CD4⁺/C-X-C motif receptor 5 (CXCR5⁺)/Programmed cell death protein 1 (PD-1⁺), and they cooperate with B cells to induce differentiation of plasmacytes through their secretion of interleukin-21 (IL-21) (89). In other chronic illnesses like systemic sclerosis and pulmonary hypertension, circulating levels of TfH are elevated and positively correlate with severity of disease (90, 91). TfH levels are also elevated in women that experience preeclampsia relative to normotensive pregnancies (92). Since IL-21 is secreted from TfH cells, IL-21 could be a cytokine of interest in the development of hypertension. One example of this effect was exhibited by Dale et al that demonstrated IL-21 and TfH cells play a role in Ang II-induced hypertension that is associated with IL-17 production (93). Infusion of Ang II into male mice resulted in a significant increase in circulating IL-21 levels relative to wildtype mice. Furthermore, global deletion of IL-21 resulted in a significant attenuation in hypertension in response to Ang II infusion. Perhaps IL-21 is an upstream cytokine that serves as a therapeutic target for hypertension.

T Helper 22 (T_H22) is a subpopulation of T_H cells (CD4⁺) which was identified in 2009 (94–96). Th22 cells secrete interleukin-22 (IL-22) which is a proinflammatory cytokine in the interleukin-10 (IL-10) cytokine family (97). T_H22 cells have been shown to contribute to Ang II-induced hypertension and endothelial dysfunction in mice (98). Ye et al further elucidated the role of T_H22 cells in hypertension through IL-22 activation via a STAT3-mediated pathway. By administering S31–201, a STAT3 pathway inhibitor, hypertension and endothelial dysfunction were ameliorated. Infusion of IL-22 in combination with Ang II produced an exaggerated hypertensive response in mice, which was associated with increased vasoconstriction and impaired nitric oxide (NO)-dependent relaxation (99). Levels of IL-22 have also been positively correlated with blood pressure in hypertensive subjects relative to healthy subjects (98).

exTreg are a subset of T_regs that have lost the Foxp3 expression and in turn, lose their anti-inflammatory properties (100). exTregs become a T_H17 cell type, becoming proinflammatory and producing IL-17. Subjects with pulmonary hypertension who exhibit chronic hypoxia have increased numbers of exTregs-T_H17 cells, suggesting that this switch might occur in a hypoxia-dependent mechanism (101). To our knowledge, there have been no reports about exTregs in arterial hypertension; however, that does not exclude the potential role exTregs could be playing in hypertension. In fact, exTregs have been identified in humans (identified as CD16⁺ CD56⁺ cytotoxic CD4⁺ T cells) (102). Given their role in inflammatory states such as arthritis, it may only be a matter of time before a role for exTregs in hypertension has been identified.

Novel Dendritic Cell Populations:

Axl⁺ Siglec6⁺ dendritic cells (AS DC) are pro-inflammatory DCs that produce cytokines such as IL-1β, IL-6, TNF-α, and IL-23 (103). In human hypertensives, there is a 3-fold increase in the number of AS DCs in the circulation relative to normotensive controls (104). Furthermore, human monocytes were co-cultured with human aortic endothelial cells (HAECs) and exposed to increasing levels of mechanical stress to replicate hypertensive stimuli. When exposed to 10% endothelial stretch, there was a greater number of AS DCs present, which could be attenuated by exposure to a growth arrest specific-6 (GAS6) siRNA. This demonstrated activation of the Axl receptor via GAS6 in order for cells to become proinflammatory and produce the mentioned cytokines and IsoLGs. To demonstrate the pro-hypertensive effects of AS DC in vivo, mice were treated with an Axl antagonist exhibited an attenuated response to Ang II infusion, which corresponded with a reduction in the number of AS DCs in renal and aortic tissue.

Myeloid DC (mDCs) are a subset of peripheral DCs that are characterized by CD11c⁺ expression and are proinflammatory (105). They are elevated in adolescencts with hypertension compared to normotensive control subjects (106). In a small cohort of subjects with and without PE, mDCs were significantly elevated in patients with PE versus control subjects (107). These levels were significantly negatively correlated with levels of vascular endothelial growth factor (VEGF) in preeclamptic women, linking mDCs with abnormal expression of VEGF.

Regulatory DCs (DC_regs) are anti-inflammatory cells that play a role in suppressing T cells. They express low levels of co-stimulatory markers, produce anti-inflammatory cytokines, promote tolerance and induces T_regs (108). Similarly to myeloid DCs, there is some evidence showing DC_regs play a role in pregnancy-induced hypertension. In an animal model of PE induced via arginine vasopression (AVP) infusion, DC_reg administration attenuated pregnancy-induced increases in blood pressure and proteinuria while improving average pup weight (109). This transfer of DC_regs also reduced circulating levels of IL-17 and IFN-γ in the maternal circulation as well as reduced levels of IL-17 in the placental tissue. To our knowledge, there are no reports of DC_regs protecting against arterial hypertension.

Plasmacytoid DCs (pDCs) are also peripheral DCs that play a critical role in orchestrating both innate and adaptive immune responses, with a significant role in T cell responses. They have been shown to be significantly lower in preeclamptic women versus normotensive controls (107). Characterized by pro-inflammatory cytokines, these cells are increased in Goldblatt hypertension in both male and female mice (110). When these mice were exposed to renovascular hypertension (2 Kidney 1 Clip hypertension) and treated with anti-mPDCA antibodies every other day for one week (500ug/mouse), both male and female mice exhibited blunted Goldblatt hypertension. This attenuation in blood pressure was associated with a significant reduction in cytokine and chemokine profiles in both sexes relative to sex-matched hypertensive controls. This work highlights the role of pDCs in the progression of hypertension in both male and female mice.

Granulocytes:

Neutrophils are the first responders in the immune system and are known for their ability of phagocytose, degranulate, and generate neutrophil extracellular traps (NETs) in a process called NETosis. In terms of hypertension, there are reports of higher number of neutrophils in hypertensive subjects (111, 112). These patients exhibit elevated superoxide anion release relative to normotensives, which is thought to be a part of the NETs. In a recent study, NETosis has been shown to cause both endothelium and end-organ damage (113). Utilizing NETosis-deficient mice (protein-arginine deiminase-r, Padi4^−/−), Krishnan et al reported that knockout mice treated with Ang II exhibited attenuated hypertension and reduced inflammation. Similarly, Chrysanthopoulou et al demonstrated that Ang II infusion in mice causes release of NETs with essential hypertension in a ROS-dependent manner (114). It has also been reported that IsoLGs are involved with neutrophils and NETs, which can be blocked by IsoLG scavenger (2-hydroxybenzylamine) in target tissues like kidney and aorta of Ang II-treated mice (115).

Myeloid-derived suppressor cells (MDSC) are immature, immunosuppressive cells that are identified by CD11b⁺Gr1⁺ (CD11b⁺ glucocorticoid receptor-1). They produce cytokines such as TGF-β and IL-10 to promote T_reg differentiation, and they produce NO and hydrogen peroxide (H₂O₂), which inhibits T cell activation (116). Shah et al. demonstrated that MDSCs accumulate in the kidneys and spleens of hypertensive mice in three different experimental models of hypertension (117). Despite these cells increasing in frequency during hypertension, they are still immunosuppressive in nature because pharmacological depletion of MDSCs exacerbated the hypertensive response to Ang II. The authors demonstrated that inhibition of MDSCs via anti-Gr1 administration (0.25mg/mouse in 200uL PBS every other day) resulted in an increase in T cell number and function, thereby exacerbating hypertension. In microbiota studies, MDSCs were shown to be increased with hypertension with a greater increase in the group that received microbiota transfer versus germfree mice, potentially linking the gut microbiota and MDSC regulation (118); however, further investigations are necessary to understand the connection between MDSCs and other immune subsets in hypertension.

Kidney cells as APCs:

The development of new spatial transcriptomic profiling and single-cell RNA sequencing techniques have highlighted that kidney cells can express immune cell markers, demonstrating they can serve as antigen presenting cells (APC). Podocytes are specialized epithelial cells on the outer surface of the glomerular capillaries that, along with endothelial cells and glomerular basement membranes, are part of the glomerular filtration barrier. The foot processes of podocytes are necessary for the selective permeability of the glomerular filtration barrier to help ensure that essential molecules remain in the blood. In the context of hypertension, chronically increased glomerular capillary pressure leads to impaired mechanotransduction, contributing to podocyte damage. Aside from the physiological role of podocytes to assist in filtrating, podocytes can serve as APCs to activate T cells in inflammatory conditions (119). In glomerular disease states, podocytes can act as immunocompetent cells, where stimulation with inflammatory cytokines can result in MHC-I and -II expression on podocyte surfaces. Presentation of these MHCs on podocytes caused differentiation and activation of T cells that lead to podocyte injury and apoptosis, ultimately contributing to nephropathy. Podocyte-specific MHC class II-deficient mice are resistant to glomerular basement membrane nephritis, indicating that immune properties of podocytes are necessary for T cell activation (120). Podocytes also express CD86, which is required for T cell activation. Human podocytes and glomerular endothelial cells also express MHC classes (120). In accordance with endothelial cells, it has recently been demonstrated that both male and female primary renal endothelial cells regulate MHC I and II in a sex-dependent manner in response to increased mechanical stretch (121). Moreover, both male and female primary renal endothelial cells also regulate key co-stimulatory molecules including CD80, CD86, and cluster of differentiation 70 (CD70) (121). Tubular epithelial cells (TEC) can also produce pro-inflammatory cytokines and chemokines via interaction with immune cells. Specifically, TECs have been shown to produce cytokines such as interferon-alpha (IFN-α) (122), B cell-activating factor (123), and CX3C motif receptor 1 (CX3CR1) (124). These are found in glomerular pathologies; however, there is limited evidence on the connection between immunocompetent kidney cells and the development of hypertension. One example of the contribution of renal TECs to inflammatory mechanisms was demonstrated by Thomas et al. (125). Male C57Bl/6 mice exhibited elevations in TEC interleukin-18 (IL-18) expression in response to DOCA salt-induced hypertension, and deletion of IL18^−/− in non-bone marrow-derived cells protected these animals from hypertension. The potential immunoregulatory role of non-hematopoietic renal cells could be a link between the development of hypertension and the immune system.

NOVEL INSIGHTS INTO THE ROLE OF IMMUNE CELLS AND THE GUT MICROBIOTA IN HYPERTENSION-RELATED INTERORGAN CROSSTALK

While the kidney has long been considered central to the regulation of blood pressure, the important contribution of ‘non-classical’, extrarenal mechanisms is becoming more evident. With immune cells serving as the mobile units of our bodies’ surveillance system, their function and location make them poised to serve as the bridge between the multiple organs that have been shown to participate in the development of hypertension.

One such extrarenal mechanism that is gaining recognition for its contribution to many chronic diseases, to include cardiovascular disease and hypertension, is the gut microbiota. Though the term gut microbiota is largely associated with the bacterial organisms that reside in the gastrointestinal tract, it is also comprised of virus, fungi, archaea, and protozoa, and exist on multiple sites of colonization across the body beyond the intestines, to include the skin, respiratory tract, and the urogenital tract. Our bodies rely on the gut microbiota for the appropriate execution of many critical host physiological functions (126), like proper digestion, nutrition, and energy metabolism. The gut microbiota is also key for maintaining the gastrointestinal (GI) barrier and mucosal layers, importantly establishing the physical, first line defense while also preserving the ability to easily move necessary nutrients, electrolytes, and bacteria-derived metabolites to the host. With the gut microbiota being intricately involved in multiple host processes, it is therefore expected that gut microbiota perturbations give rise to many different pathological conditions.

Outside of the bone marrow, lymphatics, and spleen, the gut is one of the top immunogenic organs harboring immune cells, especially lymphocytes (127). With the use of germ-free animals, it has long been shown that the gut microbiota is critical for proper immune system development and function, where the loss of gut microbiota resulted in reduced immune cell counts, impaired CD4⁺, CD8⁺, and T_reg function, and impaired B cell antibody production (128, 129). Demonstrating proof-of-principle, conventionalization of germ-free animals expanded and restored T cell populations, indicating a dynamic immunological response to microbial gut flora (130). The involvement of the gut microbiota in the development and maintenance of the immune system (131) may occur either through direct resident gut immune cell interaction or through the indirect actions of bacteria-derived metabolites once released systemically. Changes to both intestinal barrier permeability and the accessibility of microbial metabolites appear to modulate these gut-immune interactions in cardiovascular disease (CVD) (132).

One such group of beneficial microbiota-related metabolites are short-chain fatty acids (SCFAs), like propionate, butyrate, and acetate, all of which are derived from the bacterial fermentation of dietary fiber (133). SCFAs have been shown to maintain gut barrier function my promoting proper tight junction protein expression in an immune-dependent manner (134), but they also exert anti-inflammatory effects directly on innate and adaptive immune cells through SCFA receptors (135, 136). Furthermore, the favorable effects of the SCFA propionate on vascular dysfunction and hypertension rely on an increase in T_regs and corresponding decrease in effector memory T cells and T_H17 cells (137). Primary and secondary bile acids (BAs), generated through coordinated functions of the liver and microbiota, are another class of gut-derived metabolites demonstrated to affect immunity. Conjugated bile acids, which require microbiota-dependent modifications prior to systemic release and host effects carried out through nuclear receptors like the farsenoid X receptor (FXR), have been found to inversely associate with blood pressure (138). There is ample evidence suggesting these protective effects occur by dampening immunological responses by increasing anti-inflammatory IL-10-dependent M2 macrophage polarization and T_regs regulatory cells, while suppressing inflammatory T_H1 and T_H17 cells (139–141). However, perhaps the most studied microbial metabolite in CVD is trimethylamine N-oxide (TMAO). Conversely to SCFAs and BAs, TMAO is associated with increased risk for CVD and mortality (142, 143), especially in subjects with reduced kidney function (144), given the role of the kidney in TMAO clearance. Therefore, TMAO has been shown to promote immune activation and vascular inflammation via elevations in TNF-α and IL-1β, but reductions in IL-10 (145), as well as increase platelet hyperresponsiveness and the potential for pro-thrombotic events (146). This relationship between the gut microbiota and its metabolites with the immune system can then exert potentiated effects on distant organs like the kidney, heart, and brain, contributing to a multisystems disorder.

Specifically in terms of hypertension and the kidney, there is mounting evidence for modulation of the immune system in a gut microbiota-dependent manner. High salt-induced elevations in blood pressure depleted Lactobacillus murinus in the gut and concomitantly increased pro-inflammatory T_H17 cells in the lamina propria of the small intestine (75). Daily gavage and the restoration of L. murinus normalized blood pressure and reduced intestinal T_H17 cells. Similarly reflected in humans, a two-week dietary high salt challenge significantly increased circulating T_H17 cells which was associated with a loss of fecal Lactobacillus. Moreover, mice fed a high salt diet also exhibited increased intestinal inflammation, with greater presence of CD11c⁺ myeloid cells containing IsoLG-protein adducts (147). Fecal transfer of the microbiota from these high salt-fed mice resulted in the transfer of intestinal inflammation and hypertension to germ-free animals. Demonstrating that the gut microbiota is causative to hypertension and its associated renal damage via immunological mechanisms, fecal transplantation from hypertensive Dahl Salt-Sensitive rat donors worsened salt-sensitive hypertension, renal injury, and CD3⁺ T cell-specific renal infiltration in disease-resistant recipients (148). Further evidence supportive of this gut-immune-kidney relationship was shown more recently where microbial colonization of germ-free mice exacerbated hypertension and kidney damage, resulted in a deficiency in SCFAs, and an induction of pro-inflammatory T_H17 cells (118).

Given the intricate relationship between high blood pressure and the heart, the gut microbiota and the immune system have also been implicated in hypertension-associated cardiovascular disease. Patients with acute coronary syndrome, coronary artery disease, heart failure, and ischemic heart disease all exhibit perturbations in gut microbiome composition (149, 150). Pathogenic bacteria are present and elevated in individuals with chronic heart failure, which has been specifically associated with significantly higher in-hospital mortality rates (151). Patients with heart failure also exhibit reductions in specific bacteria known to contribute to SCFA production, namely those with butyrate-producing potential (152). These effects associated with a detrimental loss in butyrate is thought to be related to its anti-inflammatory properties and its promotion of T_reg differentiation and generation (153). Interestingly, heart failure is also known to contribute to multiple gastrointestinal manifestations, which may be due to poor microcirculation and gastrointestinal hypoperfusion. These changes to gut function and increased permeability cause gut edema and systemic translocation of bacteria-derived molecules, as well as bacteria themselves (154), altogether promoting the systemic immune activation that is characteristic of heart failure (155).

Individuals who have experienced ischemic stroke also similarly report gastrointestinal complications like intestinal barrier breakdown, increased gut permeability, and gut dysmotility, accompanied by robust microbiota composition changes (156). They also present with opportunistic bacteria, poorer neurological outcomes, and higher mortality rates (157). Demonstrating a causative role for these gut microbiota alterations, transplantation of dysbiotic microbiota from post-stroke mice into germ-free mice resulted in larger infarct volumes, functional impairments, and neuroinflammation marked by massive increases in IFN-γ and IL-17 (158). Importantly, through in vivo cell tracking experiments, intestinal lymphocytes were found to migrate to the ischemic brain, supporting the ‘leaky gut’ phenotype and the translocation of inflammatory immune cells.

As depictured in Figure 3, these data altogether suggest that the relationship of the gut microbiota and the immune system is certainly multidirectional with organs like the kidney, heart, and brain, and the alterations to these pathways are very likely patterns that repeat across various organ systems.

Figure 3: Interorgan Interplay of the Gut, Kidney and the Immune System in the Development of Hypertension and End-Organ Injury.

The gut microbiota and its metabolic end-products participate in the modulation of immune cell activation and function, ultimately influencing hypertensive end-organ damage to the kidneys, heart, and brain. These microbiota-related mechanisms include the gut bacteria themselves, short-chain fatty acids (SCFAs), bile acids, and the trimethylamine-N-oxide (TMAO) pathway.

CLINICAL IMPLICATIONS AND PERSPECTIVES.

Findings from murine models of hypertension and correlations in human studies have contributed to the recognition that inflammation and immunity are importantly participate in the development of hypertension and renal injury. We have discussed both innate and adaptive immune cells that regulate the development of blood pressure and renal injury through both pro- and anti-inflammatory signaling mechanisms. Recently, novel renal stromal and immune cell types have been identified contributing to the complex pathogenesis of renal inflammation and hypertension, giving fresh targets for novel therapeutic approaches. Clinically, the ideal therapy would be cell-specific to prevent inflammatory signaling, reduce renal inflammation, damage, and blood pressure, without global immunosuppression. These novel therapies could target one specific cell type to modulate pro-inflammatory or anti-inflammatory actions of renal stromal and innate and adaptive immune cells. However, the translational and therapeutic challenge is designing targeted molecules, through drug delivery systems such as target nanoparticles, that would alter specific immune responses without leaving the subject immunocompromised. Further research studies, specifically translational studies elucidating cell-specific, or organ-specific targets are needed to elucidate these signaling mechanisms in patients with renal injury and hypertension.