Critical role of Interleukin 21 and T follicular helper cells in hypertension and vascular dysfunction.

T and B cells have been implicated in hypertension, but the mechanisms by which they produce a coordinated response is unknown. T follicular helper (Tfh) cells that produce interleukin 21 (IL21) promote germinal center (GC) B cell responses leading to immunoglobulin (Ig) production. Here we investigate the role of IL21 and Tfh cells in hypertension. In response to angiotensin (Ang) II-induced hypertension, T cell IL21 production is increased, and Il21-/- mice develop blunted hypertension, attenuated vascular end-organ damage, and decreased interleukin 17A (IL17A) and interferon gamma production. Tfh-like cells and GC B cells accumulate in the aorta and plasma IgG1 is increased in hypertensive WT but not Il21-/-mice. Furthermore, Tfh cell deficient mice develop blunted hypertension and vascular hypertrophy in response to Ang II infusion. Importantly, IL21 neutralization reduces blood pressure (BP) and reverses endothelial dysfunction and vascular inflammation. Moreover, recombinant IL21 impairs endothelium-dependent relaxation ex vivo and decreases nitric oxide production from cultured endothelial cells. Finally, we show in humans that peripheral blood T cell production of IL21 correlates with systolic BP and IL17A production. These data suggest that IL21 may be a novel therapeutic target for the treatment of hypertension and its micro- and macrovascular complications.

LNK deficiency promotes acute aortic dissection and rupture.

Aortic dissection (AD) is a life-threatening vascular disease with limited treatment strategies. Here, we show that loss of the GWAS-identified SH2B3 gene, encoding lymphocyte adaptor protein LNK, markedly increases susceptibility to acute AD and rupture in response to angiotensin (Ang) II infusion. As early as day 3 following Ang II infusion, prior to the development of AD, Lnk-/- aortas display altered mechanical properties, increased elastin breaks, collagen thinning, enhanced neutrophil accumulation, and increased MMP-9 activity compared with WT mice. Adoptive transfer of Lnk-/- leukocytes into Rag1-/- mice induces AD and rupture in response to Ang II, demonstrating that LNK deficiency in hematopoietic cells plays a key role in this disease. Interestingly, treatment with doxycycline prevents the early accumulation of aortic neutrophils and significantly reduces the incidence of AD and rupture. PrediXcan analysis in a biobank of more than 23,000 individuals reveals that decreased expression of SH2B3 is significantly associated with increased frequency of AD-related phenotypes (odds ratio 0.81). Thus, we identified a role for LNK in the pathology of AD in experimental animals and humans and describe a new model that can be used to inform both inherited and acquired forms of this disease.

Hypertension and increased endothelial mechanical stretch promote monocyte differentiation and activation: roles of STAT3, interleukin 6 and hydrogen peroxide.

Aims: Monocytes play an important role in hypertension. Circulating monocytes in humans exist as classical, intermediate, and non-classical forms. Monocyte differentiation can be influenced by the endothelium, which in turn is activated in hypertension by mechanical stretch. We sought to examine the role of increased endothelial stretch and hypertension on monocyte phenotype and function. Methods and results: Human monocytes were cultured with confluent human aortic endothelial cells undergoing either 5% or 10% cyclical stretch. We also characterized circulating monocytes in normotensive and hypertensive humans. In addition, we quantified accumulation of activated monocytes and monocyte-derived cells in aortas and kidneys of mice with Angiotensin II-induced hypertension. Increased endothelial stretch enhanced monocyte conversion to CD14++CD16+ intermediate monocytes and monocytes bearing the CD209 marker and markedly stimulated monocyte mRNA expression of interleukin (IL)-6, IL-1ß, IL-23, chemokine (C-C motif) ligand 4, and tumour necrosis factor α. STAT3 in monocytes was activated by increased endothelial stretch. Inhibition of STAT3, neutralization of IL-6 and scavenging of hydrogen peroxide prevented formation of intermediate monocytes in response to increased endothelial stretch. We also found evidence that nitric oxide (NO) inhibits formation of intermediate monocytes and STAT3 activation. In vivo studies demonstrated that humans with hypertension have increased intermediate and non-classical monocytes and that intermediate monocytes demonstrate evidence of STAT3 activation. Mice with experimental hypertension exhibit increased aortic and renal infiltration of monocytes, dendritic cells, and macrophages with activated STAT3. Conclusions: These findings provide insight into how monocytes are activated by the vascular endothelium during hypertension. This is likely in part due to a loss of NO signalling and increased release of IL-6 and hydrogen peroxide by the dysfunctional endothelium and a parallel increase in STAT activation in adjacent monocytes. Interventions to enhance bioavailable NO, reduce IL-6 or hydrogen peroxide production or to inhibit STAT3 may have anti-inflammatory roles in hypertension and related conditions.

Local production of tenascin-C acts as a trigger for monocyte/macrophage recruitment that provokes cardiac dysfunction.

Aims: Tenascin-C (TNC) is an endogenous danger signal molecule strongly associated with inflammatory diseases and with poor outcome in patients with cardiomyopathies. Its function within pathological cardiac tissue during pressure overload remains poorly understood. Methods and results: We showed that TNC accumulates after 1 week of transverse aortic constriction (TAC) in the heart of 12-week-old male mice. By cross bone marrow transplantation experiments, we determined that TNC deposition relied on cardiac cells and not on haematopoietic cells. The expression of TNC induced by TAC, or by administration of a recombinant lentivector coding for TNC, triggered a pro-inflammatory cardiac microenvironment, monocyte/macrophage (MO/MΦ) accumulation, and systolic dysfunction. TNC modified macrophage polarization towards the pro-inflammatory phenotype and stimulated RhoA/Rho-associated protein kinase (ROCK) pathways to promote mesenchymal to amoeboid transition that enhanced macrophage migration into fibrillar collagen matrices. The amplification of inflammation and MO/MΦ recruitment by TNC was abrogated by genetic invalidation of TNC in knockout mice. These mice showed less ventricular remodelling and an improved cardiac function after TAC as compared with wild-type mice. Conclusions: By promoting a pro-inflammatory microenvironment and macrophage migration, TNC appears to be a key factor to enable the MO/MΦ accumulation within fibrotic hearts leading to cardiac dysfunction. As TNC is highly expressed during inflammation and sparsely during the steady state, its inhibition could be a promising therapeutic strategy to control inflammation and immune cell infiltration in heart disease.

A salt-sensing kinase in T lymphocytes, SGK1, drives hypertension and hypertensive end-organ damage.

We previously showed that angiotensin II (Ang II) increases T cell production of IL-17A, and that mice deficient in IL-17A have blunted hypertension and attenuated renal and vascular dysfunction. It was recently shown that salt enhances IL-17A production from CD4+ T cells via a serum- and glucocorticoid-regulated kinase 1-dependent (SGK1-dependent) pathway. Thus, we tested the hypothesis that SGK1 signaling in T cells promotes hypertension and contributes to end-organ damage. We show that loss of T cell SGK1 results in a blunted hypertensive response to Ang II infusion by 25 mmHg. Importantly, renal and vascular inflammation is abrogated in these mice compared with control mice. Furthermore, mice lacking T cell SGK1 are protected from Ang II-induced endothelial dysfunction and renal injury. Loss of T cell SGK1 also blunts blood pressure and vascular inflammation in response to deoxycorticosterone acetate-salt (DOCA-salt) hypertension. Finally, we demonstrate that the Na+-K+-2Cl- cotransporter 1 (NKCC1) is upregulated in Th17 cells and is necessary for the salt-induced increase in SGK1 and the IL-23 receptor. These studies demonstrate that T cell SGK1 and NKCC1 may be novel therapeutic targets for the treatment of hypertension and identify a potentially new mechanism by which salt contributes to hypertension.

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension.

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

CD4+ T cells promote the transition from hypertrophy to heart failure during chronic pressure overload.

BACKGROUND: The mechanisms by which the heart adapts to chronic pressure overload, producing compensated hypertrophy and eventually heart failure (HF), are still not well defined. We aimed to investigate the involvement of T cells in the progression to HF using a transverse aortic constriction (TAC) model. METHODS AND RESULTS: Chronic HF was associated with accumulation of T lymphocytes and activated/effector CD4(+) T cells within cardiac tissue. After TAC, enlarged heart mediastinal draining lymph nodes showed a high density of both CD4(+) and CD8(+) T-cell subsets. To investigate the role of T cells in HF, TAC was performed on mice deficient for recombination activating gene 2 expression (RAG2KO) lacking B and T lymphocytes. Compared with wild-type TAC mice, RAG2KO mice did not develop cardiac dilation and showed improved contractile function and blunted adverse remodeling. Reconstitution of the T-cell compartment into RAG2KO mice before TAC enhanced contractile dysfunction, fibrosis, collagen accumulation, and cross-linking. To determine the involvement of a specific T-cell subset, we performed TAC on mice lacking CD4(+) (MHCIIKO) and CD8(+) T-cell subsets (CD8KO). In contrast to CD8KO mice, MHCIIKO mice did not develop ventricular dilation and dysfunction. MHCIIKO mice also displayed very low fibrosis, collagen accumulation, and cross-linking within cardiac tissue. Interestingly, mice with transgenic CD4(+) T-cell receptor specific for ovalbumin failed to develop HF and adverse remodeling. CONCLUSIONS: These results demonstrate for the first time a crucial role of CD4(+) T cells and specific antigen recognition in the progression from compensated cardiac hypertrophy to HF.