From Rags to Riches: Moving Beyond RAG1 in Studies of Hypertension

The Greek philosopher Heraclitus said, “Change is the only constant in life.” We are now learning that this is unfortunately also true when working with genetically modified animals. In 2007, we demonstrated that mice deficient in functional T and B lymphocytes, recombinase activating gene 1 knock out (Rag1^−/−) mice, were protected from the full development of hypertension and hypertensive end-organ damage,¹ supporting earlier foundational work from Okuda, Grollman, and Svendsen linking the immune system to hypertension.^{2, 3} Others subsequently recapitulated this in Rag1^−/− mice, Rag1^−/− rats, and in severe combined immunodeficient (SCID) mice that also lack T and B lymphocytes (Table 1A).^{4, 5} In 2017, Kathryn Sandberg, whose lab had previously observed the same finding, published that circa 2015, the Rag1^−/− mice had changed and were no longer resistant to angiotensin II-dependent hypertension.⁶ In this issue of Hypertension, Seniuk et. al provide data suggesting that the change may have occurred even earlier (i.e. around 2009), at least in some Rag1^−/− colonies.⁷ There are 3 plausible explanations for this finding: 1) the immune system has no role in hypertension, 2) the Rag1^−/− mice have changed, and 3) varying environmental effects profoundly influence the phenotype of these mice.

Table 1.

Lessons on hypertension from immune targeted animal models

1st author/Journal/PMID	Year	Animal Model	Immune cell/Main finding
A. RAG 1 related models (negative studies are highlighted in gray):
Guzik TJ. Exp Med; PMID: 17875676	2007	Rag1−/− mouse	T cells are increased and causal in 14d Ang II (490ng/kg/min) and DOCA-salt hypertension
Uchida HA, Atherosclerosis PMID: 20362292	2010	Rag1−/− mouse	No significant difference in 28d Ang II (1000ng/kg/min) induced hypertension between WT (−48; to 160±4mmHg) and RAG1−/− (−31; to 144±4mmHg)
Marvar, PJ, Circ Res PMID 20558826	2010	Rag1−/− mouse	T cells mediate norepinephrine-induced hypertension
Vital, SA Microcirculation PMID: 21044218	2010	Rag1−/− mouse	Reduced BP responses and leukocyte adhesion in RAG1−/− mice; other models e.g.RANTES−/−;Psel−/− studied
Senchenkova EY, Hypertension, PMID: 21911709	2011	Rag1−/−; CD4−/−; CD8−/− mice	No protection from Ang II (1000 ng/kg/min) induced hypertension but protected from light-dye induced thrombosis
Marvar PJ,Biol Psychiatry, PMID: 22361077	2012	Rag1−/− mouse	T cells mediate stress induced increases of blood pressure
Mattson DL, Am J Physiol PMID: 23364523	2013	Rag1−/− rat	RAG1 deletion attenuates salt sensitive hypertension in rats
Pollow DP, Hypertension PMID: 24890822	2014	Rag1−/− mouse	Male but not female T cells mediate hypertension in Ang II infusion
Ji, H, Hypertension PMID: 24935938	2014	Rag1−/− mouse	Male but not female T cells confers Ang II 200/490 ng/kg/min) hypertension in RAG1−/− mice
Trott,, DS. Hypertension PMID: 25259750	2014	Rag1−/− CD4, MHCIIko and CD8ko mice	RAG-1−/− X OT1 mice have reduced hypertension in response to ang II. Adoptive transfer of CD8+ T cells, but not CD4+ T cells to RAG-1−/− mice restores hypertension.
Wu J, J Clin Invest PMID: 26595812	2014	Rag1−/− mouse	T cells are important and causal for hypertension and vascular fibrosis/stiffening
Mian MO, J Hypertension PMID: 26630215	2016	Rag1−/−mouse	RAG1 deficiency does not protect from hypertension, but scruffy T cell transfer lowers blood pressure.
Batchu SN, ATVB, PMID: 27365404	2016	Rag1−/− mouse	RAG-1−/− protects from late BP increase (5–6 wk) not early; Axl is critical for T cell survival in hypertensive vascular remodelling
Ji H, Hypertension PMID 28438904	2017	Rag1−/− mouse	RAG1−/− mice protected from Ang II hypertension before 2014 are no longer protected due to renal AT1R binding
Wade B, Am J Physiol, PMID: 29118017	2018	Rag1−/− rat	RAG1−/− mice protected from severe salt sensitive hypertension and renal damage
Abais-Battad JM, AM J Physiol; PMID:29537860	2018	Rag1−/− rat	RAG1−/− mice protected from protein induced hypertension and renal damage
Nhu Dinh Q; FASEB J; 32(suppl1): 718.14	2018	Rag1−/− mouse	T cells are required for aldosterone induced hypertension and end-organ damage
Senchenkova EY, Hypertension, PMID: 30739537	2019	Rag1−/−; IL6−/− mice	RAG1−/− not protected from Ang II hypertension RAG1 -Ang II SBP-122.1±10 vs 123.1±3mmHg in WT-Ang II mice. IL-6−/− protected from Ang II BP increase.
Pollow DP, Am J Physiol, PMID: 31099612	2019	Rag1−/− mouse	RAG1−/− protection of Ang II increase of BP is reversed by post-menopausal T cells
Lu X, Circ Res, PMID: 31630621	2019	Rag1−/− mouse	Myeloid cell A20 suppresses DC activation and thereby mitigates T-cell-dependent blood pressure elevation.
Seniuk A, Hypertension, 2020, in press	2020	Rag1−/− mouse	RAG1−/− not protected from Ang II hypertension at any dose as early as 2009
B. Beyond RAG-1– selected examples
De Ciuceis C, ATVB 16100037	2005	Op/Op mice	Mice lacking m-CSF and macrophages are resistant to development of hypertension, vascular remodelling and dysfunction in response to ang II
Madhur MS, Hypertension PMID: 20038749	2010	Il17a−/− mouse	IL-17A is essential in hypertension and vascular dysfunction.
Crowley SD, Hypertension PMID: 20147609	2010	SCID mouse	Immune deficiency attenuates hypertension
Vinh B., Circulation PMID: 21126972	2010	B7−/− mouse; abatacept	Classical antigen presentation involving B7/CD28 is involved in hypertension and vascular dysfunction
Barhoumi T, Hypertension PMID 21263125	2011	WT mouse	T regulatory cells prevent ang II-induced hypertension and vascular injury
Wenzel P., Circulation PMID: 21875910	2011	LysM+(iDTR) mouse	Lysozyme M-positive monocytes mediate Ang II-induced arterial hypertension and vascular dysfunction.
Nguyen H, PMID 23263331	2013	WT mouse	IL-17A causes inhibitory phosphorylation of eNOS via a Rho kinase. IL-17A administration causes hypertension.
Kossmann S., ATVB PMID: 23520167	2013	Ifnγ−/−; Tbx21−/− mice	Role of monocytes and NK cells in driving Ang II-induced HTN and vascular dysfunction
Amador CA, Hypertension PMID: 24420551	2014	IL-17A mAb	IL17mAb reduces blood pressure and fibrosis
Kirabo A., J Clin Invest PMID: 25244096	2013	DC transfer	DCs are essential for hypertension and isolevuglandins are essential for neoantigen generation
Trott, DW, Hypertension PMID: 25259750	2014	CD8−/−; OT1xRAG-1−/− mice	Oligoclonal CD8+ T cells play a critical role in the development of hypertension.
Zimmerman MA, Am J Renal Physiol, PMID 25503730	2015	Sprague-Dawley rat	Sex differences in renal accumulation of proinflammatory - vs. anti-inflammatory T cells
Carnevale D, Immunity PMID: 25517614	2014	Plgf−/− mouse	The angiogenic factor PlGF mediates a neuroimmune interaction in the spleen to allow the onset of hypertension.
Shah KH, Circ Res PMID: 26294657	2015	Depletion or adoptive transfer of MDSCs	Myeloid derived suppressor cells limit inflammation and blood pressure increase in hypertensive mouse models.
Chen C, Hypertension PMID: 26351030	2015	Baffr−/− mouse	B lymphocytes contribute to hypertension
Saleh MA, J Clin Invest PMID: 26595812	2015	Lnk−/−; Ifnγ−/− mice	Lymphocyte adaptor protein LNK deficiency exacerbates hypertension and end-organ inflammation.
Santisteban MM, Circ Res; PMID: 25963715	2015	SHR/WKY rat	Bone marrow derived cells are essential for BP increase and neuroinflammation in hypertension.
Krishnan SM, B J P PMID:26103560	2016	Asc−/− mouse	Inflammasome is essential in experimental hypertension
Xiao L., Hypertension PMID: 26156232	2015	Renal denervation mouse	Renal denervation prevents immune cell activation and renal inflammation in Ang II-induced hypertension.
Mikolajczyk TP, FASEB J PMID: 26873938	2016	Rantes−/−	The chemokine RANTES is essential for regulation of vascular inflammation and dysfunction in HTN
Itani HA, Circ Res PMID: 26988069	2016	Cd70−/− mouse	CD70 exacerbates blood pressure elevation and renal damage in response to repeated hypertensive stimuli.
Norlander A, Hypertension PMID: 27141060	2016	Il17A−/− mouse	Interleukin-17A Regulates Renal Sodium Transporters and Renal Injury in Angiotensin II-Induced Hypertension.
Harwani SC, Circ Res PMID: 27660287	2016	SHR (rat)	Describes novel α7-nicotinic receptor activation of macrophages in pre-hypertensive SHR
Carnevale D, Nat Commun PMID: 27676657	2016	α7nAChR−/− mouse; Vagotomy	A cholinergic-sympathetic pathway primes immunity in hypertension regulates Ang II induced BP increase.
Singh MV, J Allergy Clin Immunol. PMID: 28093217	2017	SHR (rat)	Implicates the transcription factor RORγt and the cytokine IL17F in genesis of hypertension in SHR
Saleh MA JACC BasicTranslSci PMID: 28280792	2016	IL-17A mAb	IL-17A but not IL-17F is causal in hypertension and effect can be targeted by pharmacological inhibition
Norlander AE, PMID 28679951	2017	T cell Sgk1−/− mouse	The salt sensing kinase in T cells contributes to hypertension caused by either salt feeding or ang II
Caillon A, Circulation PMID: 28330983	2017	Tcrδ−/− mouse	γδ T Cells Mediate Angiotensin II-Induced Hypertension and Vascular Injury.
Wilck N, Nature PMID: 29143823	2017	Microbiota treated mice	Salt-responsive gut commensal modulates TH17 axis and disease.
Hevia D, Hypertension PMID: 29378857	2018	Cd11c−/− mouse	CD11c ablation prevents hypertension
Dale BL; JCI Insight PMID: 31013256	2019	Il21−/− mouse	Critical role of Interleukin 21 and T follicular helper cells in hypertension and vascular dysfunction.
Raikwar N; Am J Physiol, Heart and Circ Physiol PMID: 31172810	2019	SHR (rat)	Renal denervation prevents renal infiltration of CD161+ T cells and macrophages in pre-hypertensive spontaneously hypertensive rats,
Van Beusecum, Hypertension PMID: 31280647	2019	DC Sgk1−/− mouse	Deletion of SGK1 in dendritic cells reduces renal inflammation and hypertension in a salt sensitive model

The first hypothesis – that the immune system has no role in hypertension – is disproven by a myriad of published studies. Spurred by the earlier work on mice and rats with a complete deficiency of the adaptive immune system, we and numerous independent laboratories have now collected a rich dataset that clearly defines specific immune cell subsets, cytokines, and chemokines that contribute positively or negatively to hypertension pathophysiology (Table 1B). Genetic deletion or pharmacological inhibition of the cytokine interleukin 17A (produced by Th17 and γ T cells) decreases blood pressure and renal and vascular end-organ damage. Likewise, Singh et al. provided evidence that IL17F contributes to the development of hypertension in spontaneously hypertensive rats. Genetic deletion or pharmacological inhibition of IL-21 (produced by Th17 and Tfh cells) also results in blunted hypertension and reduced end-organ damage. Th17 cells have been shown to be salt-sensitive and produce more IL-17A in response to increased extracellular sodium via the salt-sensing kinase, SGK1. We demonstrated that deletion of SGK1 specifically in T lymphocytes is protective against angiotensin II and deoxycorticosterone-acetate (DOCA) salt hypertension. We also found that the Th1/Tc1 cytokine, IFNγ, promotes hypertension. Caillon et al. demonstrated that loss of gamma delta (γδ) T cells protects against hypertension and vascular injury. Studies from Barhoumi et al. demonstrated a protective role of Treg cells in hypertension, and Zimmerman et al. demonstrated that differences in Treg cells and other T cell subsets may underlie sex differences in hypertension. Elegant work from Drs. Daniela Carnevale and Giuseppe Lembo demonstrate the importance of neural modulation of immune function to blood pressure control and suggests that selective splenic denervation may represent a novel therapeutic strategy for resistant hypertension. In keeping with this, renal denervation, which shows some promise for resistant hypertension treatment, is associated with decreased renal accumulation of T cells and macrophages.

Dendritic cells (DCs) and similar antigen presenting cells (APCs) are essential for T cell activation by processing and presenting antigenic peptides and by providing T cell co-stimulation. DCs have been demonstrated to play a major role in hypertension. In 2010, we showed that T cells require a second stimulus via interaction with B7 ligands on APCs to cause hypertension. Blockade of the B7/CD28 T-cell co-stimulation axis by treatment with CTLA4-Ig or genetic deletion of B7 ligands prevents both angiotensin II and DOCA-salt-induced hypertension. More recently, we demonstrated that CD70 on antigen presenting cells is involved in the formation of memory T cells that contribute to hypertension in response to repeated hypertensive stimuli. We also demonstrated that DCs from hypertensive mice drive memory T cell proliferation and prime naïve mice to develop hypertension in response to a subpressor dose of angiotensin II. While low dose angiotensin II induced severe hypertension in wild type mice that received DCs from angiotensin II infused mice, it failed to increase blood pressure in Rag1^−/− mice, indicating that T cells are required for the DC-mediated priming of hypertension. Similarly, Lu et al. recently demonstrated that the ubiquitin-editing protein A20 in DCs regulates blood pressure by limiting renal T cell activation. Crossing mice with DC specific heterozygous deletion of A20 with Rag1^−/− mice abrogates the hypertensive response to low dose angiotensin II, suggesting that DC A20 mediates hypertension in a T cell-dependent manner. Moreover, we found that hypertension may be antigen-mediated. Hypertension is associated with oxidative modification of self-proteins by products of lipid peroxidation known as isolevuglandins (isoLGs). Kirabo et al. showed that DCs accumulate and present isoLG-protein adducts to T cells during hypertension and exhibit increased expression of co-stimulatory molecules such as CD80 and CD86. IsoLGs also induce increased production of the proinflammatory cytokines IL-1β, IL-6, and IL-23 by DCs, which activates T cells to proliferate and produce pro-hypertensive cytokines such as IL-17A and IFNγ. Furthermore, we recently showed that SGK1 also plays an important role in DCs by modulating NADPH oxidase activation and isoLG formation in these cells.

In contrast to immunodeficient Rag1^−/− mice, mice lacking the lymphocyte adaptor molecule LNK/Sh2b3 have increased hematopoiesis, larger spleens, and more circulating and resident immune cells in tissues. We demonstrated that these mice have an exaggerated hypertensive response to angiotensin II, and that bone marrow from Lnk^−/− mice transplanted into wild type mice completely recapitulates the hypertensive response. Of note, LNK is a negative regulator of cytokine signaling and cell proliferation and was discovered in multiple human genome wide association studies to be associated with hypertension and other cardiovascular disorders. Similarly, Santisteban et al. transplanted bone marrow from spontaneously hypertensive rats into normal Wistar-Kyoto rats and showed that this raised blood pressure as well as central and peripheral inflammation. Thus, a multitude of studies, many using animal models completely independent of Rag1 deficiency, have confirmed a critical role for the immune system in hypertension and begun to elucidate an intricate network of innate and adaptive immune cells that orchestrate blood pressure control and contribute causally to hypertensive end-organ damage.

The second and third possibilities – that the Rag1^−/− mice have changed their phenotype or are highly influenced by environmental factors – are both highly plausible and potentially related. The use of genetically manipulated animals for research is in its relative infancy, and we have yet to determine the long-term consequences of deleting a gene in a whole animal, particularly a gene as critical as Rag1 for immune function. As said by the fictional character, Dr. Ian Malcolm, in the sci-fi classic Jurassic Park, “Life finds a way.” Given the built-in complexity and redundancies in the immune system, it is possible that over generations, the immune system finds a way to compensate. It has been shown that Rag1^−/− mice have an expanded population of natural killer cells, which could produce many of the same cytokines normally produced by T cells that contribute to blood pressure elevations and end-organ damage. Although usually buried in lab notebooks and not published in peer-reviewed journals, the loss of animal phenotypes is certainly not a new phenomenon as many investigators are often left scratching their heads about what has changed in their mouse colony to result in the loss of a previously robust phenotype. A change in trainees, laboratory location, housing temperature, season, diet, and the microbiome are all proposed as possible contributing factors. As an example of the importance of the microbiome to hypertension, Karbach et al. demonstrated that germ-free mice are protected from angiotensin II-induced renal and vascular inflammation and hypertension.⁸ For these reasons and others, perhaps it is time to focus less on animal models and more on human studies.

Clinical evidence:

There is a rich source of data in humans that the immune system is associated with hypertension. Of course, most human studies are correlative as performing manipulations of the immune system in relatively healthy people with isolated hypertension is still too premature and risky. However, we can obtain clues from patients with other autoimmune and inflammatory disorders. Autoimmune diseases in humans including psoriasis, systemic lupus erythematosus and rheumatoid arthritis are associated with increased T cell production of IL-17A and/or IFNγ and an increased risk of hypertension.⁹ Herrera et al. showed that administration of the immunosuppressive drug mycophenolate mofetil reduced hypertension in patients with psoriasis and rheumatoid arthritis.¹⁰ Interestingly and providing some evidence of causality, von Stebut et al. demonstrated in a randomized, double-blind, placebo-controlled trial that in patients with moderate to severe plaque psoriasis, anti-IL-17A treatment with secukinumab improved endothelial function as assessed by flow mediated dilation.¹¹ Blood pressures during treatment were not reported in this study. People living with HIV exhibit increased inflammation associated with increased circulating cytokines and accumulation of CD8⁺ T cells compared to HIV-negative controls and are at a heightened risk for developing hypertension.^12–14 Youn et al. demonstrated that hypertension in humans is associated with an accumulation of senescent CD8⁺ T cells in the blood that produce pro-inflammatory and cytotoxic factors such as perforin and granzyme B.¹⁵ We showed that isoLGs accumulate in monocytes and dendritic cells of humans with hypertension.⁷ Another link between the innate immune response and hypertension in humans comes from Furman et al. who demonstrated that high expression of specific inflammasome gene modules, including constitutive expression of IL-1β, was associated with hypertension and arterial stiffness in older individuals.¹⁶ Consistent with these findings, our RNA sequence (RNA-seq) analysis of monocytes isolated from the peripheral blood of normotensive and hypertensive humans showed that hypertension is associated with changes in the expression of at least 60 genes, many of which are involved in pro-inflammatory pathways, including IL-1β signaling and T cell activation.¹⁷ Furthermore, we recently showed that T cell production of IL-21 correlates with IL-17A production and systolic blood pressure in humans.¹⁸

In conclusion, although our initial findings with the Rag1^−/− mice may have been a fleeting phenotype, it fortuitously spurred a whole field of research on immune mechanisms of hypertension in both animal models and humans that has expanded well beyond the Rag1^−/− mouse and weaves a story that stands completely on its own. It is no longer a question of “does the immune system play an important role in hypertension”, but rather how and why, and which immune cells and cytokines should we be targeting to treat hypertensive end-organ damage. While mice provide an easily manipulatable platform to answer basic questions, the differences in mouse and human immune systems and the more complex questions that now need to be answered will require moving into human cells, human organoids, human biobanks and electronic health records, and finally human clinical trials to answer. Cutting-edge single cell transcriptomic and proteomic approaches to deeply immunophenotype human hypertension and identify novel inflammatory biomarkers and targets will pave the way for a future of immunomodulation, as opposed to immunosuppression, to treat humans suffering from this number one risk factor for global morbidity and mortality.