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A subset of interleukin (IL)-17A-producing γδ T cells called γδT17 cells may contribute to progression of hypertension. γδT17 cell development is in part dependent upon IL-23 receptor (IL-23R) stimulation. We hypothesized that angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular injury would be blunted in Il23r knock-in (Il23r

The COVID‐19 pandemic extended all around the world causing millions of deaths. In addition to acute respiratory distress syndrome, many patients with severe COVID‐19 develop thromboembolic complications associated to multiorgan failure and death. Here, we review evidence for the contribution of neutrophils, platelets, and extracellular vesicles (EVs) to the thromboinflammatory process in COVID‐19. We discuss how the immune system, influenced by pro‐inflammatory molecules, EVs, and neutrophil extracellular traps (NETs), can be caught out in patients with severe outcomes. We highlight how the deficient regulation of the innate immune system favors platelet activation and induces a vicious cycle amplifying an immunothrombogenic environment associated with platelet/NET interactions. In light of these considerations, we discuss potential therapeutic strategies underlining the modulation of purinergic signaling as an interesting target.

Introduction: We have demonstrated that the P2X7 receptor (P2RX7), which activates immune cells via binding of extracellular adenosine triphosphate, plays a role in hypertension and vascular injury. Angiotensin (Ang) II-induced hypertension, vascular injury and perivascular infiltration of activated CD8 + T cells were attenuated by P2rx7 knockout or P2RX7 pharmacologic blockade. However, whether P2RX7 is involved in Ang II-induced cardiac dysfunction and hypertrophy remains unknown. Hypothesis: We hypothesized that Ang II-induced left ventricular (LV) dysfunction and hypertrophy will be blunted equally by P2rx7 knockout and P2RX7 antagonism. Methods: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7 -/- mice were infused or not with Ang II (1000 ng/kg/min) for 14 days. A second group of WT mice infused with Ang II was infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle for 14 days. Cardiac LV function and remodeling were determined by ultrasound, and hypertrophic markers in cardiac ventricles by reverse transcription-quantitative PCR (RT-qPCR). Results: Ang II-induced increase in LV mass/body weight (6.3±0.2 vs 4.2±0.2 mg/g, P P P2rx7 -/- mice (7.3±0.5 mg/g and 20.2±3.1%, P Nppa /ribosomal protein S16 [ Rps16 ], 6.8±2.3 vs 1±0.1, P Acta1 / Rps16 , 6.3±1.3 vs 1±0.2, P P2rx7 -/- mice ( Nppa / Rps16 : 13.5±2.4 and Acta1 / Rps16 : 11.9±2.4), but not in AZ10606120-treated mice ( Nppa / Rps16 : 5.5±0.7 and Acta1 / Rps16 : 5.6±0.8). Conclusion: Ang II-induced LV hypertrophy and dysfunction were exaggerated by P2rx7 knockout but were not affected by P2RX7 pharmacologic blockade.

Introduction: The innate-like γδ T cells play a role in angiotensin II (AngII)-induced hypertension, vascular injury and T cell activation in perivascular adipose tissue (PVAT). Hypothesis: We hypothesized that single cell RNA sequencing (scRNA-seq) will reveal γδ T cell subpopulations in PVAT involved in hypertension, vascular injury and T cell activation. Methods: Male C57BL/6J mice were infused SC or not with 490 ng/kg/min AngII for 14 days (n=3). Hypertension was confirmed by tail cuff blood pressure measurement. Mesenteric vessels (MV) with PVAT were collected, lymph nodes removed, single cell suspension obtained and labeled with hashtag antibodies, T cells isolated by fluorescence-activated cell sorting, the 6 samples pooled in one tube, and scRNA libraries prepared with a Chromium Next GEM Single Cell 3’ Reagent Kit. Sequencing was done on an Illumina Novaseq 6000, and data analysed using Cell Ranger pipeline and Seurat tools. Cell subpopulations were validated by flow cytometry. Results: ScRNA-seq yielded 5,030 cells with 137,990 reads/cell and identified 11 T cell clusters. Subclustering of T cells expressing the T cell receptor (TCR) δ constant chain revealed 3 δ natural killer T (δNKT) (δNKT0, δNKT1 and activated δNKT cells) and 3 γδ T cell subpopulations ( Trgc1 low effector memory, TCR δ variable 4 + and apoptotic γδ T cells). AngII increased >2-fold the frequency of activated δNKT cells, and decreased by 74% δNKT0 cells. Gene expression profiling revealed that activated δNKT and δNKT0 cells could be identified using unique markers, Cd28 and Sell , respectively. Flow cytometry showed that TCRδ + CD28 + Sell - T cells were increased (393±57.2 vs 227±44.4 cells/MV-PVAT) and TCRδ + CD28 - Sell + T cells decreased (21.6±4.1 vs 45.3±4.10 cells/MV-PVAT) in MV-PVAT of AngII vs sham-treated mice. Conclusion: This study identified an activated δNKT cell subpopulation in MV-PVAT that may play a role in AngII-induced hypertension, vascular injury and T cell activation.

Introduction: Innate and adaptive immune cells contribute to hypertension and end-organ damage. High blood pressure (BP) causes cardiovascular injury and the release of damage-associated molecule patterns such as adenosine triphosphate (ATP). ATP can bind to the purinergic receptor P2X7 (P2RX7) on innate immune cells triggering interleukin-1β release, which drives further immune activation. Hypothesis: We hypothesized that P2rx7 knockout or P2RX7 antagonism would blunt angiotensin II (AngII)-induced BP elevation and cardiovascular injury through decreased immune activation. Methods: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7 -/- mice were infused or not with AngII (1000 ng/kg/min) for 14 days. A second group of AngII-infused WT mice was also infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle. BP was determined by telemetry, plasma ATP using a bioluminescence assay, mesenteric artery function using pressurized myography, cardiac left ventricle (LV) function and mass by ultrasound and activated immune T cell infiltration in aortic perivascular adipose tissue (PVAT) by flow cytometry. Results: AngII increased plasma ATP in WT mice (4.4±1.2 vs 2.0±0.9 μM, P P2rx7 deficiency (164±3 vs 176±2 mm Hg, P P P P P2rx7 -/- (FS: 20.2±3.1% and LVmass/BW: 7.2±0.5 mg/g, P P P2rx7 -/- or AZ10606120-treated mice. AngII increased CD69 + CD8 + T cell infiltration in aortic PVAT of WT (60±16 vs 16±3 cells/aortic PVAT, P P2rx7 -/- or AZ10606120-treated mice. Conclusion: P2rx7 knockout or antagonism attenuates AngII-induced BP elevation, vascular injury, and infiltration of activated CD8 + T cells into aortic PVAT. P2rx7 knockout exacerbated AngII-induced cardiac dysfunction and hypertrophy, whereas P2RX7 antagonism did not.

The processes of activation, extravasation, and migration of immune cells to a site are early and essential steps in the induction of an acute inflammatory response. These events are an essential part of the inflammatory cascade, which involves multiple regulatory steps. Using a murine air pouch model of inflammation with LPS as an inflammation inducer, we demonstrate that isoenzymes of the neuraminidase family (NEU1, 3, and 4) play essential roles in these processes by acting as positive or negative regulators of leukocyte infiltration. In genetically knocked-out (KO) mice for different NEU genes (Neu1 KO, Neu3 KO, Neu4 KO, and Neu3/4 double KO mice) with LPS-induced air pouch inflammation, leukocytes at the site of inflammation were counted, and the inflamed tissue was analyzed using immunohistochemistry. Our data show that leukocyte recruitment was decreased in NEU1- and NEU3-deficient mice, while it was increased in NEU4-deficient animals. Consistent with these results, systemic as well as pouch exudate levels of pro-inflammatory cytokines were reduced in Neu1 and increased in Neu4 KO mice. Pharmacological inhibitors specific for NEU1, NEU3, and NEU4 isoforms also affected leukocyte recruitment. Together our data demonstrate that NEU isoenzymes have distinct-and even opposing-effects on leukocyte recruitment, and therefore warrant further investigation to determine their mechanisms and importance as regulators of the inflammatory cascade.

Background: The P2X7 receptor (P2RX7) recognizes damage associated molecule patterns such as adenosine triphosphate (ATP), and triggers the activation of immune cells. Elevated plasma ATP levels have been observed in hypertensive patients, providing a potential mechanism for P2RX7 activation. Additionally, a hypomorphic polymorphism for P2X7 is correlated with a decreased risk for essential hypertension in Chinese post-menopausal women. However, it is unknown whether P2RX7 activation contributes to angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular damage. We hypothesized that P2rx7 knockout would blunt Ang II-induced BP elevation, vascular injury, and infiltration of activated immune T cells into perivascular adipose tissue (PVAT). Methods: Ten-to-12-week-old male C57BL/6J male wild-type (WT) and P2rx7 -/- mice were infused or not with Ang II (1000ng/kg/min) for 14 days. BP was determined by telemetry, mesenteric artery function and remodeling using pressurized myography, aortic stiffening by ultrasound and infiltration of activated immune T cells in aortic PVAT by flow cytometry. Results: Ang II-infused P2rx7 -/- mice display a reduced systolic BP (164±3 vs. 176±2 mm Hg, P P P + T cells in aortic PVAT (60±16 vs 16±3 cells/aortic PVAT, P P2rx7 -/- mice (6.4±1.4 vs 5.5±1.1 m/s and 27±7 vs 16±3 cells/aortic PVAT). In addition, the frequency of IFN-γ producing CD8 + T cells in the spleen of Ang II-treated WT mice increased (2.6±0.2% vs 1.2±0.2%), which did not occur in P2rx7 -/- mice (1.7±0.3% vs 1.7±0.2%). Ang II-infusion induced mesenteric artery endothelial dysfunction in WT mice (61±7 vs 83±4% relaxation response to acetylcholine, P P2rx7 -/- mice (89±3 vs 90±3%). Conclusion: P2rx7 knockout attenuates Ang II-induced hypertension, vascular injury, and infiltration of activated CD8 + T cells into aortic PVAT.

Background: Accumulating evidence has revealed that coronavirus disease 2019 (COVID-19) patients may be complicated with myocardial injury during hospitalization. However, data regarding persistent cardiac involvement in patients who recovered from COVID-19 are limited. Our goal is to further explore the sustained impact of COVID-19 during follow-up, focusing on the cardiac involvement in the recovered patients.Methods: In this prospective observational follow-up study, we enrolled a total of 40 COVID-19 patients (20 with and 20 without cardiac injury during hospitalization) who were discharged from Zhongnan Hospital of Wuhan University for more than 6 months, and 27 patients (13 with and 14 without cardiac injury during hospitalization) were finally included in the analysis. Clinical information including self-reported symptoms, medications, laboratory findings, Short Form 36-item scores, 6-min walk test, clinical events, electrocardiogram assessment, echocardiography measurement, and cardiac magnetic resonance imaging was collected and analyzed.Results: Among 27 patients finally included, none of patients reported any obvious cardiopulmonary symptoms at the 6-month follow-up. There were no statistically significant differences in terms of the quality of life and exercise capacity between the patients with and without cardiac injury. No significant abnormalities were detected in electrocardiogram manifestations in both groups, except for nonspecific ST-T changes, premature beats, sinus tachycardia/bradycardia, PR interval prolongation, and bundle-branch block. All patients showed normal cardiac structure and function, without any statistical differences between patients with and without cardiac injury by echocardiography. Compared with patients without cardiac injury, patients with cardiac injury exhibited a significantly higher positive proportion in late gadolinium enhancement sequences [7/13 (53.8%) vs. 1/14 (7.1%), p = 0.013], accompanied by the elevation of circulating ST2 level [median (interquartile range) = 16.6 (12.1, 22.5) vs. 12.5 (9.5, 16.7); p = 0.044]. Patients with cardiac injury presented higher levels of aspartate aminotransferase, creatinine, high-sensitivity troponin I, lactate dehydrogenase, and N-terminal pro–B-type natriuretic peptide than those without cardiac injury, although these indexes were within the normal range for all recovered patients at the 6-month follow-up. Among patients with cardiac injury, patients with positive late gadolinium enhancement presented higher cardiac biomarker (high-sensitivity troponin I) and inflammatory factor (high-sensitivity C-reactive protein) on admission than the late gadolinium enhancement–negative subgroup.Conclusions: Our preliminary 6-month follow-up study with a limited number of patients revealed persistent cardiac involvement in 29.6% (8/27) of recovered patients from COVID-19 after discharge. Patients with cardiac injury during hospitalization were more prone to develop cardiac fibrosis during their recovery. Among patients with cardiac injury, those with relatively higher cardiac biomarkers and inflammatory factors on admission appeared more likely to have cardiac involvement in the convalescence phase.

Background The risk that COVID-19 patients develop critical illness that can be fatal depends on their age and immune status and may also be affected by comorbidities like hypertension. The goal of this study was to develop models that predict outcome using parameters collected at admission to the hospital. Methods and Results This is a retrospective single-center cohort study of COVID-19 patients at the Seventh Hospital of Wuhan City, China. Forty-three demographic, clinical and laboratory parameters collected at admission plus discharge/death status, days from COVID-19 symptoms onset and days of hospitalization were analyzed. From 157 patients, 120 were discharged and 37 died. Pearson correlations showed that hypertension and systolic blood pressure (SBP) were associated with death and respiratory distress parameters. A penalized logistic regression model efficiently predicts the probability of death with 13 of 43 variables. A regularized Cox regression model predicts the probability of survival with 7 of above 13 variables. SBP but not hypertension was a covariate in both mortality and survival prediction models. SBP was elevated in deceased compared to discharged COVID-19 patients. Conclusions Using an unbiased approach, we developed models predicting outcome of COVID-19 patients based on data available at hospital admission. This can contribute to evidence-based risk prediction and appropriate decision-making at hospital triage to provide the most appropriate care and ensure the best patient outcome. High SBP, a cause of end-organ damage and an important comorbid factor, was identified as a covariate in both mortality and survival prediction models., Graphical Abstract Graphical Abstract

(1) Background: Chronic increases in blood flow, as in cardiovascular diseases, induce outward arterial remodeling. Thrombospondin-1 (TSP-1) is known to interact with matrix proteins and immune cell-surface receptors, but its contribution to flow-mediated remodeling in the microcirculation remains unknown. (2) Methods: Mesenteric arteries were ligated in vivo to generate high- (HF) and normal-flow (NF) arteries in wild-type (WT) and TSP-1-deleted mice (TSP-1−/−). After 7 days, arteries were isolated and studied ex vivo. (3) Results: Chronic increases in blood flow induced outward remodeling in WT mice (increasing diameter from 221 ± 10 to 280 ± 10 µm with 75 mmHg intraluminal pressure) without significant effect in TSP-1−/−(296 ± 18 to 303 ± 14 µm), neutropenic or adoptive bone marrow transfer mice. Four days after ligature, pro inflammatory gene expression levels (CD68, Cox2, Gp91phox, p47phox and p22phox) increased in WT HF arteries but not in TSP-1−/− mice. Perivascular neutrophil accumulation at day 4 was significantly lower in TSP-1−/− than in WT mice. (4) Conclusions: TSP-1 origin is important, indeed, circulating TSP-1 participates in vasodilation, whereas both circulating and tissue TSP-1 are involved in arterial wall thickness and diameter expansion.

Inflammatory processes have been shown to play an important role in the mechanisms involved in the pathogenesis of hypertension. Innate and adaptive immune responses participate in BP elevation and end-organ damage. Here, we discuss recent studies focusing on novel inflammatory and immune mechanisms that play roles in BP elevation. Different subpopulations of cells involved in innate and adaptive immune responses, such as dendritic cells, monocytes/macrophages and NK cells, on the one hand, and B and T lymphocytes, on the other, contribute to the vascular and kidney injury in hypertension. Unconventional innate-like T cells such as γδ T cells also participate in hypertensive mechanisms by priming both innate and adaptive immune cells, contributing to trigger vascular inflammation and BP elevation. These cells exert their effects in part via production of various cytokines including pro-inflammatory IFN-γ and IL-17 and anti-inflammatory IL-10. The present review summarizes some of these immune mechanisms that participate in the pathophysiology of hypertension. LINKED ARTICLES: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.

Endothelium-derived endothelin (ET)-1 has been implicated in the development of hypertension and end-organ damage, but its exact role remains unclear. We have shown that tamoxifen-inducible endothelium-restricted human ET-1 overexpressing (ieET-1) mice exhibited blood pressure rise after a 3-week induction in an ET type A (ET A ) receptor-dependent manner, in absence of vascular and renal injury. It is unknown whether long-term ET-1 overexpression results in sustained blood pressure elevation and vascular and renal injury. Adult male ieET-1 and control tamoxifen-inducible endothelium-restricted Cre recombinase (ieCre) mice were induced with tamoxifen and 2.5 months later, were treated with or without the ET A receptor blocker atrasentan for 2 weeks. Three-month induction of endothelial human ET-1 overexpression increased blood pressure ( P P P P Col1A1 and Col3A1 expression, and perivascular adipose tissue oxidative stress ( P P P + ) and myeloid-derived suppressive cell (CD11b + Gr-1 + ) renal infiltration ( P P P A receptors.

Aims Matrix metalloproteinases (MMPs) have been implicated in the development of hypertension in animal models and humans. Mmp2 deletion did not change Ang II-induced blood pressure (BP) rise. However, whether Mmp2 knockout affects angiotensin (Ang) II-induced vascular injury has not been tested. We sought to determine whether Mmp2 knockout will prevent Ang II-induced vascular injury. Methods and results A fourteen-day Ang II infusion (1000 ng/kg/min, SC) increased systolic BP, decreased vasodilatory responses to acetylcholine, induced mesenteric artery (MA) hypertrophic remodelling, and enhanced MA stiffness in wild-type (WT) mice. Ang II enhanced aortic media and perivascular reactive oxygen species generation, aortic vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 expression, perivascular monocyte/macrophage and T cell infiltration, and the fraction of spleen activated CD4+CD69+ and CD8+CD69+ T cells, and Ly-6Chi monocytes. Study of intracellular signalling showed that Ang II increased phosphorylation of epidermal growth factor receptor and extracellular-signal-regulated kinase 1/2 in vascular smooth muscle cells isolated from WT mice. All these effects were reduced or prevented by Mmp2 knockout, except for systolic BP elevation. Ang II increased Mmp2 expression in immune cells infiltrating the aorta and perivascular fat. Bone marrow (BM) transplantation experiments revealed that in absence of MMP2 in immune cells, Ang II-induced BP elevation was decreased, and that when MMP2 was deficient in either immune or vascular cells, Ang II-induced endothelial dysfunction was blunted. Conclusions Mmp2 knockout impaired Ang II-induced vascular injury but not BP elevation. BM transplantation revealed a role for immune cells in Ang II-induced BP elevation, and for both vascular and immune cell MMP2 in Ang II-induced endothelial dysfunction.

Background: Innate antigen-presenting cells and adaptive immune T cells have been implicated in the development of hypertension. However, the T-lymphocyte subsets involved in the pathophysiology of hypertension remain unclear. A small subset of innate-like T cells expressing the γδ T cell receptor (TCR) rather than the αβ TCR could play a role in the initiation of the immune response in hypertension. We aimed to determine whether angiotensin (Ang) II caused kinetic changes in γδ T cells; deficiency in γδ T cells blunted Ang II-induced hypertension, vascular injury, and T-cell activation; and γδ T cells are associated with human hypertension. Methods: Male C57BL/6 wild-type and Tcrδ −/− mice, which are devoid of γδ T cells, or wild-type mice injected IP with control isotype IgG or γδ T cell-depleting antibodies, were infused or not with Ang II for 3, 7, or 14 days. T-cell profiling was determined by flow cytometry, systolic blood pressure (SBP) by telemetry, and mesentery artery endothelial function by pressurized myography. TCR γ constant region gene expression levels and clinical data of a whole blood gene expression microarray study, including normotensive and hypertensive subjects, were used to demonstrate an association between γδ T cells and SBP. Results: Seven- and 14-day Ang II infusion increased γδ T-cell numbers and activation in the spleen of wild-type mice ( P P P Tcrδ −/− mice ( P P Tcrδ −/− mice ( P R 2 =0.12, P -6 ). Conclusions: γδ T cells mediate Ang II-induced SBP elevation, vascular injury, and T-cell activation in mice. γδ T cells might contribute to the development of hypertension in humans.

Objectives: Both innate and adaptive immune cells have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subset of “innate-like” T lymphocytes, expressing the γδ T cell receptor (TCR) rather than the αβ TCR, plays a key role in hypertension and vascular injury. We demonstrated an increased number and activation (CD69 + ) of αβ and γδ T cells during the development of hypertension caused by angiotensin (Ang) II infusion, and that deficiency in γδ T cells prevented Ang II-induced hypertension, resistance artery endothelial dysfunction and spleen T-cell activation in mice. We hypothesized that γδ T cells mediate activation of other T cells in hypertension. Methods: C57BL/6 male mice were infused with Ang II (490 ng/kg/min, SC) for 7 and 14 days (n=5-7). All mice were 14-15 week-old at the end of the study. Spleen T cell profile was determined by flow cytometry. γδ and αβ T cells were isolated using magnetic beads from peripheral lymph nodes and spleen from C57BL/6 male mice treated or not with Ang II for 14 days. αβ T cells were cultured alone or with γδ T cells (5:1) in presence of anti-CD3 antibodies plus or minus Ang II, and αβ T cell phenotype was evaluated by flow cytometry. Results: Close correlations were demonstrated between the number (#) of activated CD69 + γδ T cells and CD4 + CD69 + T cells (r 2 =0.74, P + CD69 + T cells (r 2 =0.64, P + CD69 + γδ T cells and CD4 + CD69 + T cells (r 2 =0.76, P + CD69 + T cells (r 2 =0.65, P In vitro , Ang II increased the fraction of CD69 + αβ T cells when αβ T cells were co-cultured with γδ T cells isolated from control mice (% of αβ T cells: 19.9±2.9 vs. 16.4±2.6, P + (% of CD69 + αβ T cells: 35.7±2.7 vs. 18.9±1.5, P + αβ T cells (% of CCR6 + αβ T cells: 27.6±2.4 vs. 19.2±3.5, P Conclusions: These results suggest that γδ T cells mediate activation of αβ T cells in Ang II-induced hypertension. Targeting γδ T cells may contribute to reduce the low-grade inflammation found in hypertension.

Introduction: Both innate and adaptive immune cells (such as T lymphocytes) have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subpopulation of T cells considered "innate-like", expressing the γδ T-cell receptor (TCR) rather than the much more frequent αβ TCR, plays a key role in hypertension and vascular injury. The number and activation of γδ T cells were increased after 7 days of angiotensin (Ang) II infusion, and absence of γδ T cells prevented the development of hypertension, endothelial dysfunction of resistance arteries, and activation of CD4 + and CD8 + T cells in mice infused with Ang II for 14 days. γδ T cells can be subdivided according to the TCR variant (V) subtype that is generally specific for a tissue. A subpopulation of γδ T cells in the lungs and skin that are Vγ6 + and produce interleukin (IL)-17A was shown to respond promptly to pneumococcal infection and skin inflammation. However, γδ T cell Vγ subtypes involved in hypertension are still unknown. We hypothesized that γδ T cells involved in hypertension are Vγ6 + . Methods: Eleven- to 13-week old C57BL/6J male mice were infused or not with Ang II (490 ng /kg /min, sc) for 14 days (n = 5-14), and γδ T cell Vγ subtypes were profiled by flow cytometry in the spleen, mesenteric lymph nodes (MLNs), thoracic aortic (TA), perivascular adipose tissue (PVAT) and mesenteric artery (MA) PVAT. Results: In spleen and MLNs the most abundant γδ T cell Vγ subtypes were Vγ1,2 + and Vγ4 + followed by Vγ6 + , Vγ5 + and Vγ7 + . In TA PVAT, the most abundant γδ T cell Vγ subtype was Vγ6 + followed by Vγ4 + , Vγ1,2 + , Vγ5 + and Vγ7 + . In MA PVAT, the most abundant γδ T cell Vγ subtype was Vγ6 + followed by Vγ4 + , Vγ7 + , Vγ5 + and Vγ1,2 + . Ang II infusion increased the frequency of Vγ6 + γδ T cells in the spleen (% of CD3 + T cells: 0.77±0.09 vs. 0.5±0.02, P P P =0.07). The Vγ6 + γδ T cell frequency in MLNs was unaffected by Ang II infusion. Conclusion: A differential distribution of γδ T cell Vγ subtypes was observed in lymphoid organs compared to PVAT. Vγ6 + γδ T cells may play a role in hypertension. Targeting Vγ6 + γδ T cells could be a therapeutic approach to reduce inflammation in hypertension.

To describe the important role played by innate and innate-like immunity in the pathophysiology of hypertension and vascular injury. Innate immune cells, such as neutrophils, dendritic cells, myeloid-derived suppressor cells, and monocytes/macrophages and innate lymphoid cells such as natural killer cells and unconventional T lymphocytes like γδ T cells contribute to hypertensive mechanisms by priming adaptive immune cells, leading to the triggering of vascular inflammation and blood pressure elevation or alternatively protecting against vascular injury. Specifically, monocyte/macrophages and γδ T cells seem to play a crucial role in the initiation of hypertension via regulation of adaptive immunity. Innate and innate-like immunity play a leading role in the pathophysiology of hypertension. Recent advances in this field provide us clues for future therapeutic approaches.

International audience; Ectonucleoside triphosphate diphosphohydrolase-1, the major vascular/immune ectonucleotidase, exerts anti-thrombotic and immunomodulatory actions by hydrolyzing extracellular nucleotides (danger signals). Hypertension is characterized by vascular wall remodeling, endothelial dysfunction, and immune infiltration. Here our aim was to investigate the impact of arterial hypertension on CD39 expression and activity in mice. Arterial expression of CD39 was determined by reverse transcription quantitative real-time PCR in experimental models of hypertension, including angiotensin II (AngII)-treated mice (1 mg/kg/day, 21 days), deoxycorticosterone acetate-salt mice (1% salt and uninephrectomy, 21 days), and spontaneously hypertensive rats. A decrease in CD39 expression occurred in the resistance and conductance arteries of hypertensive animals with no effect on lymphoid organs. In AngII-treated mice, a decrease in CD39 protein levels (Western blot) was corroborated by reduced arterial nucleotidase activity, as evaluated by fluorescent (etheno)-ADP hydrolysis. Moreover, serum-soluble ADPase activity, supported by CD39, was significantly decreased in AngII-treated mice. Experiments were conducted in vitro on vascular cells to determine the elements underlying this downregulation. We found that CD39 transcription was reduced by proinflammatory cytokines interleukin (IL)-1β and tumor necrosis factor alpha on vascular smooth muscle cells and by IL-6 and anti-inflammatory and profibrotic cytokine transforming growth factor beta 1 on endothelial cells. In addition, CD39 expression was downregulated by mechanical stretch on vascular cells. Arterial expression and activity of CD39 were decreased in hypertension as a result of both a proinflammatory environment and mechanical strain exerted on vascular cells. Reduced ectonucleotidase activity may alter the vascular condition, thus enhancing arterial damage, remodeling, or thrombotic events.

Dr Campbell raises the concern regarding our study on γδ T cells and hypertension1 that the dose (490 ng·kg–1·min–1) and subcutaneous route of angiotensin II infusion is too high in comparison with that required to increase blood pressure intravenously.2 There are indeed important differences in bioavailability between both routes of administration. The dose of 490 ng·kg–1·min–1 induces similar pressor responses and plasma levels in rats as intravenous angiotensin II at 20 ng·kg–1·min–1, according to Dr Campbell’s own review from a few years ago.2 He points out that high concentrations of angiotensin II delivered into the subcutaneous tissue may activate a variety of cells expressing angiotensin receptors, including T …

Objective: Both innate (monocyte/macrophages) and adaptive immune cells (T lymphocytes) have been shown to play a role in the development of vascular injury in hypertension. Recently, we demonstrated that a small subset of “innate-like” T lymphocytes, expressing the γ/δ T cell receptor (TCR) rather than the αβ TCR, plays a key role in hypertension and vascular injury. We demonstrated an increased number and activation (CD69 + ) of γδ T cells during the development of hypertension caused by angiotensin (Ang) II infusion, and that deficiency in γδ T cells prevented Ang II-induced hypertension, resistance artery endothelial dysfunction and spleen T-cell activation in mice. We hypothesized that γδ T cells mediate activation of other T cells in hypertension. Method and Results: Fourteen to 15-week old male C57BL/6 wild-type (WT) mice were infused with Ang II (490 ng/kg/min, SC) for 3, 7 and 14 days (n=5-7) and spleen T cell profile was determined by flow cytometry. A correlation was demonstrated between the frequency (FREQ) and the number (#) of activated CD69 + γδ T cells and CD4 + CD69 + T cells (FREQ: r=0.41, P P + CD69 + T cells (FREQ: r=0.36, P P + γδ T cells expressing CD27, a marker of interferon-γ expressing cells and a member of the T-T interaction molecules, with CD4 + CD69 + (r=0.88, P + CD69 + (r=0.81, P Conclusion: This study demonstrated an association between CD27 + CD69 + γδ T cells and activated T cells. These results suggest that γδ T cells drive activation of other T cells in Ang II-induced hypertension. Targeting γδ T cells may contribute to reduce inflammation in hypertension.

Introduction: Endothelium-derived endothelin (ET)-1 has been implicated in hypertension and renal disease but the mechanisms are complex and remain unclear. We have shown that tamoxifen-inducible endothelium-restricted human ET-1 overexpressing (ieET-1) mice exhibited BP rise after 3 weeks of induction in an ET type A receptor (ET A R)-dependent manner, in absence of renal injury. It is unknown whether long-term exposure to ET-1 overexpression results in sustained BP elevation and renal injury. Methods: Adult male ieET-1 and control tamoxifen-inducible endothelium-restricted Cre recombinase (ieCre) mice were treated with tamoxifen (1 mg/kg/day, SC) for 5 days and 2.5 months later were treated or not with an ET A R blocker, atrasentan (10 mg/kg/day, PO) for 2 weeks. Blood pressure (BP) by telemetry, renal artery flow (RAF) by ultrasonography, immune cell infiltration by flow cytometry, kidney injury molecule (KIM)-1 expression by immunofluorescence, 24h urinary albumin by ELISA and creatinine by alkaline picrate method were determined at the end of the study. Results: Induction of ET-1 overexpression for 3 months resulted in greater systolic BP (135±4 vs 114±2 mmHg, P P + cells/kidney, P + Gr-1 + cells/kidney, P + (23.2±1.8 vs 7.5±1.6 % of CD45 + cells, P - , 5.7±0.8 vs 3.2±0.6 % of CD45 - cells, P P P Conclusions: Long-term exposure to endothelial ET-1 overexpression caused sustained BP elevation and renal injury via ET A R.

Background: Vascular injury is an early manifestation of hypertension. microRNAs (miRNAs) play an important role in cardiovascular disease, but their implication in vascular injury remains unclear. Using small and total RNA sequencing, we identified in murine mesenteric arteries (MAs) a conserved angiotensin (Ang) II-upregulated Dlk1-Dio3 miRNA miR-431 that correlated with blood pressure (BP), and an Ang II-downregulated BP-correlated conserved putative miR-431 target, the transcriptional factor ETS homologous factor ( Ehf ). miR-431 might be involved in vascular remodeling as Ehf regulates expression of extracellular matrix genes including alpha-1 type I collagen ( Col1a1 ) and of other Dlk1-Dio3 miRNAs. In this study, we proposed to validate the miR-431- Ehf - Col1a1 interaction in vitro and in vivo , and determine whether miR-431 inhibition antagonizes angiotensin (Ang) II-induced hypertension and vascular injury. Methods and Results: Transfection of miR-431 mimics into human aortic smooth muscle cells decreased Ehf expression (0.13±0.05 fold, P Col1a1 (1.7±0.5 fold, P Ehf (1.5±0.2 fold, P Col1a1 (0.89±0.11 fold, P Ehf siRNA transfection increased 1.2±0.1 fold Col1a1 ( P EHF 3’ UTR binding site decreased 0.51±0.01 fold ( P P Col1a1 (0.58±0.11 fold, P Ehf (2.9±0.8 fold, P P P P Conclusion and Perspectives: miR-431 and its target Ehf may act as master regulators in the pathophysiology of vascular damage in hypertension. miR-431 inhibition has the potential to serve as a novel therapeutic approach for treatment of vascular damage and hypertension.

Objective: Both innate antigen-presenting cells and the adaptive immune system have been shown to play a role in the development of hypertension. Nevertheless, the T cell subsets involved in the pathophysiology of hypertension remains unclear. There is a small subset of “innate-like” T cells expressing the γ/δ T cell receptor (TCR) rather than the α/β TCR that could play a role bridging the innate and adaptive immune systems. We previously observed that angiotensin (Ang) II caused an increase in number and activation of γ/δ T cells and Ang II-induced systolic blood pressure (SBP) rise and vascular injury were blunted in Tcr δ -/- mice, which are devoid of γ/δ T cells. In order to further characterize the role of γ/δ T cells in hypertension, we determined whether Ang II-effects would be blunted by antibody-induced depletion of γ/δ T cells. In addition, we tested whether SBP in human could be predicted by combining the expression of genes encoding TCRGC (TCR gamma constant region) and pro-inflammatory markers of γ/δ T cells in peripheral blood mononuclear cells (PBMC). Method and Results: Thirteen to 15-week old male C57BL/6 wild-type (WT) mice were infused with Ang II (490 ng/kg/min, SC) for 14 days and injected IP with anti-γ/δ TCR or control isotype antibodies 1 day before and at day 6 of Ang II infusion. Depletion of γ/δ T cells decreased SBP (147±2 vs 167 ± 3 mm Hg, P max : 90±4 vs 62 ± 8%, P P Conclusion: Antibody-induced depletion further demonstrates the role of γ/δ T cells in Ang II-induced SBP elevation and vascular injury. Prediction of SBP using PBMC gene expression of γ/δ T cells and pro-inflammatory markers suggests that γ/δ T cells contribute to the development of human hypertension.

Inflammation has been shown to play an important role in the mechanisms involved in the pathogenesis of hypertension. Accordingly, innate and adaptive immune responses participate in blood pressure elevation. Here, we describe recent immunity studies focusing on novel inflammatory mechanisms during the hypertensive process. Different subpopulations of cells involved in innate and adaptive immune responses, such as monocyte/macrophages and dendritic cells on the one hand and B and T lymphocytes on the other hand, play roles leading to vascular injury in hypertension. Innate lymphoid cells, including natural killer cells and γ/δ T cells, have recently been demonstrated to participate in hypertensive mechanisms triggering vascular inflammation. In summary, we discuss the evidence of interaction of these different inflammatory and immune components in both experimental models and in humans during the development of hypertension.

Objective: Both innate antigen presenting cells and the adaptive immune system have been shown to play a role in the development of hypertension. Nevertheless, the T cell subset involved in the pathophysiology of hypertension remains unclear. There is a small subset of “innate-like” T cells expressing gamma/delta T cell receptor (TCR) rather than the alpha/beta TCR that could play a role in bridging between the innate and adaptive immune systems. However, it is unknown whether gamma/delta T cells contribute to development of hypertension. Method/Results: Thirteen to 15 week-old male C57BL/6 wild-type and Tcrd-/- mice, which are devoid of gamma/delta T cells, were infused or not with angiotensin (Ang) II (490 ng/kg/min, SC) for 7 or 14 days (n=4-9). Telemetric blood pressure, mesenteric artery endothelial function and vascular remodeling by pressurized myography and spleen T cell profile by flow cytometry were evaluated. Fourteen days of Ang II increased systolic blood pressure (167±4 vs 125±2 mmHg, P≤0.01) in wild-type compared to control mice. The frequency of gamma/delta T cells (6±1% vs 3±1%, P≤0.05) and activated (CD69+) gamma/delta T cells (11±1% vs 7±1%) was increased after 7 days of Ang II, and 7 days later were respectively unchanged or further increased (24±2% vs 10±1%) in wild-type compared to control mice. Ang II decreased mesenteric artery relaxation responses to acetylcholine (51±5% vs 88±3%, P≤0.01) and increased media/lumen (5±1 vs 3±0%, P≤0.01) in wild-type mice compare to controls. No gamma/delta T cells were detected in Tcrd-/- treated or not with Ang II. All the above Ang II effects were abrogated in Tcrd-/- mice. Conclusion: These data suggest that gamma/delta T cells mediate Ang II-induced blood pressure rise and vascular injury. Gamma/delta T cells could be key immune cells bridging innate and adaptive immune responses during the development of hypertension.

Objective: Flow (shear stress)-mediated outward remodeling of resistance arteries is involved in collateral growth during postischemic revascularization. Besides the NO pathway the involvement of inflammatory factors and reactive oxygen species has been suggested in this remodeling although the mechanism and the sequence remains unknown. As T cells drive the inflammatory response, we investigated their role in flow-mediated remodeling. Mouse mesenteric resistance arteries (250μm internal diameter) were submitted in vivo to a chronic increase (144±18 to 239±25 μl/min) in blood flow. Arteries were collected for in vitro analysis after 1 to 7 days. Results: After 1 week, remodeling occurred in high flow (HF) arteries (diameter increased from 242±21μm to 324±20μm, n=9 per group, P Conclusion: we demonstrate for the first time the involvement of lymphocytes T cells polarization into TH17 by angiotensin II type 2 receptor in flow-mediated outward remodeling of resistance arteries.

Heme oxygenase 1 is induced by hemodynamic forces in vascular smooth muscle and endothelial cells. We investigated the involvement of heme oxygenase 1 in flow (shear stress)-dependent remodeling. Two or 14 days after ligation of mesenteric resistance arteries, vessels were isolated. In rats, at 14 days, diameter increased by 23% in high-flow arteries and decreased by 22% in low-flow arteries compared with normal flow vessels. Heme oxygenase activity inhibition using Tin-protoporphyrin abolished diameter enlargement in high-flow arteries and accentuated arterial narrowing in low-flow arteries (32% diameter decrease versus 22% in control). Two days after ligation, heme oxygenase 1 expression increased in high-flow and low-flow vessels, in association with a reduced mitochondrial aconitase activity (marker of oxidative stress) in high-flow arteries only. Inhibition of macrophage infiltration (clodronate) decreased heme oxygenase 1 induction in low-flow but not in high-flow arteries. Similarly, inhibition of NADPH oxidase activity (apocynin) decreased heme oxygenase 1 induction in low-flow but not high-flow arteries. However, dihydroethidium staining was higher in high-flow and low-flow compared with normal flow arteries. In arteries cannulated in an arteriograph, heme oxygenase 1 mRNA increased in a flow-dependent manner and was abolished by N G -nitro- l -arginine methyl ester, catalase, or mitochondrial electron transport chain inhibition. Furthermore, heme oxygenase 1 induction using cobalt-protoporphyrin restored altered high-flow remodeling in endothelial NO synthase knockout mice. Thus, in high-flow remodeling, heme oxygenase 1 induction depends on shear stress–generated NO and mitochondria-derived hydrogen peroxide. In low-flow remodeling, heme oxygenase 1 induction requires macrophage infiltration and is mediated by NADPH oxidase–derived superoxide.

The nervous system influences immune responses through the release of neural factors such as neuropeptides. Among them, the tachykinin substance P (SP) signals via the neurokinin 1 receptor (NK-1R), which is expressed by various immune cells. We thereby analyzed in this paper whether tachykinins may participate in human CD4+ Th cell polarization. We report that SP and hemokinin-1 (HK-1) upregulate IL-17A and IFN-γ production by human memory CD4+ T cells without affecting IL-4 and IL-10 production. SP and HK-1 switch non–Th17-committed CD4+ memory T cells into bona fide Th17 cells and Th1/Th17 cells. In contrast, SP and HK-1 do not modulate the polarization of naive CD4+ T cells. SP- and HK-1–induced Th17 cell generation is mediated through NK-1R and requires the presence of monocytes. SP and HK-1 trigger IL-1β, IL-6, and TNF-α production, upregulate IL-23 production, and enhance TNF-like 1A expression on monocyte surface. Neutralization experiments demonstrated that IL-1β, IL-23, and TNF-like 1A are involved in the SP- and HK-1–induced Th17 cell. The other members of the tachykinin family, neurokinins A and B, have no effect on the differentiation of naive and memory T cells. These results thereby show that SP and HK-1 are novel Th17 cell-inducing factors that may act locally on memory T cells to amplify inflammatory responses.

ObjectiveFlow (shear stress)-mediated outward remodeling of resistance arteries is involved in collateral growth during postischemic revascularization. Besides the NO pathway the involvement of inflammatory factors and reactive oxygen species has been suggested in this remodeling although the mechanism and the sequence remains unknown. As T cells drive the inflammatory response, we investigated their role in flow-mediated remodeling. Mouse mesenteric resistance arteries (250μm internal diameter) were submitted in vivo to a chronic increase (144±18 to 239±25μl/min) in blood flow. Arteries were collected for in vitro analysis after 1 to 7 days.ResultsAfter 1 week, remodeling occurred in high flow (HF) arteries (diameter increased from 242±21μm to 324±20μm, n=xx per group, P