The CD27/CD70 pathway negatively regulates visceral adipose tissue-resident Th2 cells and controls metabolic homeostasis.

Adipose tissue homeostasis relies on the interplay between several regulatory lineages, such as type 2 innate lymphoid cells (ILC2s), T helper 2 (Th2) cells, regulatory T cells, eosinophils, and type 2 macrophages. Among them, ILC2s are numerically the dominant source of type 2 cytokines and are considered as major regulators of adiposity. Despite the overlap in immune effector molecules and sensitivity to alarmins (thymic stromal lymphopoietin and interleukin-33) between ILC2s and resident memory Th2 lymphocytes, the role of the adaptive axis of type 2 immunity remains unclear. We show that mice deficient in CD27, a member of the tumor necrosis factor receptor superfamily, are more resistant to obesity and associated disorders. A comparative analysis of the CD4 compartment of both strains revealed higher numbers of fat-resident memory Th2 cells in the adipose tissue of CD27 knockout mice, which correlated with decreased programmed cell death protein 1-induced apoptosis. Our data point to a non-redundant role for Th2 lymphocytes in obesogenic conditions.

Chronic CD27-CD70 costimulation promotes type 1-specific polarization of effector Tregs.

Introduction: Most T lymphocytes, including regulatory T cells, express the CD27 costimulatory receptor in steady state conditions. There is evidence that CD27 engagement on conventional T lymphocytes favors the development of Th1 and cytotoxic responses in mice and humans, but the impact on the regulatory lineage is unknown. Methods: In this report, we examined the effect of constitutive CD27 engagement on both regulatory and conventional CD4+ T cells in vivo, in the absence of intentional antigenic stimulation. Results: Our data show that both T cell subsets polarize into type 1 Tconvs or Tregs, characterized by cell activation, cytokine production, response to IFN-γ and CXCR3-dependent migration to inflammatory sites. Transfer experiments suggest that CD27 engagement triggers Treg activation in a cell autonomous fashion. Conclusion: We conclude that CD27 may regulate the development of Th1 immunity in peripheral tissues as well as the subsequent switch of the effector response into long-term memory.

PHD2 Constrains Antitumor CD8+ T-cell Activity.

The prolyl hydroxylase domain/hypoxia-inducible factor (PHD/HIF) pathway has been implicated in a wide range of immune and inflammatory processes, including in the oxygen-deprived tumor microenvironment. To examine the effect of HIF stabilization in antitumor immunity, we deleted Phd2 selectively in T lymphocytes using the cre/lox system. We show that the deletion of PHD2 in lymphocytes resulted in enhanced regression of EG7-OVA tumors, in a HIF-1α-dependent manner. The enhanced control of neoplastic growth correlated with increased polyfunctionality of CD8+ tumor-infiltrating lymphocytes, as indicated by enhanced expression of IFNγ, TNFα, and granzyme B. Phenotypic and transcriptomic analyses pointed to a key role of glycolysis in sustaining CTL activity in the tumor bed and identified the PHD2/HIF-1 pathway as a potential target for cancer immunotherapy.

Inhaled dry powder cisplatin increases antitumour response to anti-PD1 in a murine lung cancer model.

Despite advances in targeted therapies and immunotherapy in lung cancer, chemotherapy remains the backbone of treatment in most patients at different stages of the disease. Inhaled chemotherapy is a promising strategy to target lung tumours and to limit the induced severe systemic toxicities. Cisplatin dry powder for inhalation (CIS-DPI) was tested as an innovative way to deliver cisplatin locally via the pulmonary route with minimal systemic toxicities. In vivo, CIS-DPI demonstrated a dose-dependent antiproliferative activity in the M109 orthotopic murine lung tumour model and upregulated the immune checkpoint PD-L1 on lung tumour cells. Combination of CIS-DPI with the immune checkpoint inhibitor anti-PD1 showed significantly reduced tumour size, increased the number of responders and prolonged median survival over time in comparison to the anti-PD1 monotherapy. Furthermore, the CIS-DPI and anti-PD1 combination induced an intra-tumour recruitment of conventional dendritic cells and tumour infiltrating lymphocytes, highlighting an anti-tumour immune response. This study demonstrates that combining CIS-DPI with anti-PD1 is a promising strategy to improve lung cancer therapy.

Agonistic anti-CD27 antibody ameliorates EAE by suppressing IL-17 production.

CD27/CD70 costimulation enhances T-cell survival, memory formation and Th1-cell differentiation and effector function. In addition to promoting Th1 responses, CD27 signaling has been shown to exert a negative regulatory role on IL-17 production, resulting in increased sensitivity of CD27 KO mice to EAE. By inducing EAE in full CD27 KO mice, and in a novel, T-cell specific CD27 KO mouse strain (CD4-Cre x CD27flox/flox ), we demonstrate herein that CD27 engagement by its natural ligand (CD70) suppresses IL-17 production in a cell autonomous fashion. We further show that CD27 engagement by an agonistic antibody given after EAE induction or at symptom onset similarly suppresses IL-17 production by activated CD4+ T cells infiltrating the inflamed CNS while IFN-γ production was unaffected, leading to an amelioration of inflammatory-related symptoms. These findings propose CD27 costimulation as a potential candidate for therapeutic manipulation to treat autoimmune and autoinflammatory diseases characterized by excessive IL-17 production.

The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells.

The oxygen sensor prolyl hydroxylase domain 2 (PHD2) plays an important role in cell hypoxia adaptation by regulating the stability of HIF proteins (HIF1α and HIF2α) in numerous cell types, including T lymphocytes. The role of oxygen sensor on immune cells, particularly on regulatory T cell (Treg) function, has not been fully elucidated. The purpose of our study was to evaluate the role of PHD2 in the regulation of Treg phenotype and function. We demonstrate herein that selective ablation of PHD2 expression in Treg (PHD2ΔTreg mice) leads to a spontaneous systemic inflammatory syndrome, as evidenced by weight loss, development of a rectal prolapse, splenomegaly, shortening of the colon, and elevated expression of IFN-γ in the mesenteric lymph nodes, intestine, and spleen. PHD2 deficiency in Tregs led to an increased number of activated CD4 conventional T cells expressing a Th1-like effector phenotype. Concomitantly, the expression of innate-type cytokines such as Il1b, Il12a, Il12b, and Tnfa was found to be elevated in peripheral (gut) tissues and spleen. PHD2ΔTreg mice also displayed an enhanced sensitivity to dextran sodium sulfate-induced colitis and toxoplasmosis, suggesting that PHD2-deficient Tregs did not efficiently control inflammatory response in vivo, particularly those characterized by IFN-γ production. Further analysis revealed that Treg dysregulation was largely prevented in PHD2-HIF2α (PHD2-HIF2αΔTreg mice), but not in PHD2-HIF1α (PHD2-HIF1αΔTreg mice) double KOs, suggesting an important and possibly selective role of the PHD2-HIF2α axis in the control of Treg function. Finally, the transcriptomic analysis of PHD2-deficient Tregs identified the STAT1 pathway as a target of the PHD2-HIF2α axis in regulatory T cell phenotype and in vivo function.

Adenosine Diphosphate and the P2Y13 Receptor Are Involved in the Autophagic Protection of Ex Vivo Perfused Livers From Fasted Rats: Potential Benefit for Liver Graft Preservation.

Studies on how to protect livers perfused ex vivo can help design strategies for hepatoprotection and liver graft preservation. The protection of livers isolated from 24-hour versus 18-hour starved rats has been previously attributed to autophagy, which contributes to the energy-mobilizing capacity ex vivo. Here, we explored the signaling pathways responsible for this protection. In our experimental models, 3 major signaling candidates were considered in view of their abilities to trigger autophagy: high mobility group box 1 (HMGB1), adenosine monophosphate-activated protein kinase (AMPK), and purinergic receptor P2Y13. To this end, ex vivo livers isolated from starved rats were perfused for 135 minutes, after which perfusate samples were studied for protein release and biopsies were performed for evaluating signaling protein contents. For HMGB1, no significant difference was observed between livers isolated from rats starved for 18 and 24 hours at perfusion times of both 0 and 135 minutes. The phosphorylated and total forms of AMPK, but not their ratios, were significantly higher in 24-hour fasted than in 18-hour fasted livers. However, although the level of phosphorylated AMPK increased, perfusing ex vivo 18-hour fasted livers with 1 mM 5-aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, did not protect the livers. In addition, the adenosine diphosphate (ADP; and not adenosine monophosphate [AMP]) to AMP + ADP + adenosine triphosphate ratio increased in the 24-hour starved livers compared with that in the 18-hour starved livers. Moreover, perfusing 24-hour starved livers with 0.1 mM 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde (MRS2211), a specific antagonist of the P2Y13 receptor, induced an increase in cytolysis marker levels in the perfusate samples and a decrease in the levels of autophagic marker microtubule-associated proteins 1 light chain 3 II (LC3II)/actin (and a loss of p62/actin decrease), indicating autophagy inhibition and a loss of protection. The P2Y13 receptor and ADP (a physiological activator of this receptor) are involved in the protection of ex vivo livers. Therapeutic opportunities for improving liver graft preservation through the stimulation of the ADP/P2Y13 receptor axis are further discussed.

Long-term T cell fitness and proliferation is driven by AMPK-dependent regulation of reactive oxygen species.

The AMP-activated kinase (AMPK) is a major energy sensor metabolic enzyme that is activated early during T cell immune responses but its role in the generation of effector T cells is still controversial. Using both in vitro and in vivo models of T cell proliferation, we show herein that AMPK is dispensable for early TCR signaling and short-term proliferation but required for sustained long-term T cell proliferation and effector/memory T cell survival. In particular, AMPK promoted accumulation of effector/memory T cells in competitive homeostatic proliferation settings. Transplantation of AMPK-deficient hematopoïetic cells into allogeneic host recipients led to a reduced graft-versus-host disease, further bolstering a role for AMPK in the expansion and pathogenicity of effector T cells. Mechanistically, AMPK expression enhances the mitochondrial membrane potential of T cells, limits reactive oxygen species (ROS) production, and resolves ROS-mediated toxicity. Moreover, dampening ROS production alleviates the proliferative defect of AMPK-deficient T cells, therefore indicating a role for an AMPK-mediated ROS control of T cell fitness.

Mitochondrial dysfunction, AMPK activation and peroxisomal metabolism: A coherent scenario for non-canonical 3-methylglutaconic acidurias.

The occurrence of 3-methylglutaconic aciduria (3-MGA) is a well understood phenomenon in leucine oxidation and ketogenesis disorders (primary 3-MGAs). In contrast, its genesis in non-canonical (secondary) 3-MGAs, a growing-up group of disorders encompassing more than a dozen of inherited metabolic diseases, is a mystery still remaining unresolved for three decades. To puzzle out this anthologic problem of metabolism, three clues were considered: (i) the variety of disorders suggests a common cellular target at the cross-road of metabolic and signaling pathways, (ii) the response to leucine loading test only discriminative for primary but not secondary 3-MGAs suggests these latter are disorders of extramitochondrial HMG-CoA metabolism as also attested by their failure to increase 3-hydroxyisovalerate, a mitochondrial metabolite accumulating only in primary 3-MGAs, (iii) the peroxisome is an extramitochondrial site possessing its own pool and displaying metabolism of HMG-CoA, suggesting its possible involvement in producing extramitochondrial 3-methylglutaconate (3-MG). Following these clues provides a unifying common basis to non-canonical 3-MGAs: constitutive mitochondrial dysfunction induces AMPK activation which, by inhibiting early steps in cholesterol and fatty acid syntheses, pipelines cytoplasmic acetyl-CoA to peroxisomes where a rise in HMG-CoA followed by local dehydration and hydrolysis may lead to 3-MGA yield. Additional contributors are considered, notably for 3-MGAs associated with hyperammonemia, and to a lesser extent in CLPB deficiency. Metabolic and signaling itineraries followed by the proposed scenario are essentially sketched, being provided with compelling evidence from the literature coming in their support.

HO-1 mitigates acute kidney injury and subsequent kidney-lung cross-talk.

Ischemia-reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI), which contributes to the development of chronic kidney disease (CKD). IRI-induced AKI releases proinflammatory cytokines (e.g. IL-1ß, TNF-α, IL-6) that induce a systemic inflammatory response, resulting in proinflammatory cells recruitment and remote organ damage. AKI is associated with poor outcomes, particularly when extrarenal complications or distant organ injuries occur. Acute lung injury (ALI) is a major remote organ dysfunction associated with AKI. Hence, kidney-lung cross-talk remains a clinical challenge, especially in critically ill population. The stress-responsive enzyme, heme oxygenase-1 (HO-1) is largely known to protect against renal IRI and may be preventively induced using hemin prior to renal insult. However, the use of hemin-induced HO-1 to prevent AKI-induced ALI remains poorly investigated. Mice received an intraperitoneal injection of hemin or sterile saline 1 day prior to surgery. Twenty-four hours later, mice underwent bilateral renal IRI for 26 min or sham surgery. After 4 or 24 h of reperfusion, mice were sacrificed. Hemin-induced HO-1 improved renal outcomes after IRI (i.e. fewer renal damage, renal inflammation, and oxidative stress). This protective effect was associated with a dampened systemic inflammation (i.e. IL-6 and KC). Subsequently, mitigated lung inflammation was found in hemin-treated mice (i.e. neutrophils influx and lung KC). The present study demonstrates that hemin-induced HO-1 controls the magnitude of renal IRI and the subsequent AKI-induced ALI. Therefore, targeting HO-1 represents a promising approach to prevent the impact of renal IRI on distant organs, such as lung.

Protection in a model of liver injury is parallel to energy mobilization capacity under distinct nutritional status.

OBJECTIVE: Dietary and energetic restrictions are endowed with protection against experimental injuries. However, a drop in cell energetic status under a critical threshold may prevent protection, as previously observed for livers isolated from rat donors undergoing 18-h fasting versus feeding. The aim of this study was to further explore, in the latter model, links between nutritional status, energy availability, and protection through lengthening of rat fasting to 24 h and withdrawal of energy sources from perfusions. METHODS: Energy-free perfused ex vivo livers from fed, 18-h-fasted, and 24-h-fasted rats were studied during 135 min for cytolysis (potassium, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase releases in perfusates), cell deaths (activated caspase-3 [apoptosis], LC3 II/actin and p62/actin ratios [autophagy]), glycogen stores, glucose, and lactate production. RESULTS: Cytolysis was significantly increased by 18-h and 24-h fasting versus feeding but unexpectedly the increase was less for 24-h fasting than it was for 18-h fasting. Apoptotic marker caspase 3 significantly increased under fed and 18-h fasting but not 24-h fasting conditions. Autophagic marker LC3 II/actin significantly increased during perfusion in the 24-h fasted group but neither fed nor 18-h fasted groups. Autophagic induction also was supported by a drop in the p62/actin ratio. Under perfusion with 3-methyladenine, a standard autophagy inhibitor, protection and enhanced autophagy provided by 24-h but not 18-h fasting were lost without affecting apoptosis. CONCLUSIONS: Liver protections are obviously influenced by nutritional status in a way that is parallel to hepatic energy mobilization capacities (glycogen plus autophagy) with a decreased order of protection: Fed >24-h fasted >18-h fasted >24-h fasted + 3-methyladenine livers. By showing that autophagy induction limits starvation-induced cytolysis, the present work supports the emerging view that autophagy inducers might improve health benefits of diet restriction.

Multiple Environmental Signaling Pathways Control the Differentiation of RORγt-Expressing Regulatory T Cells.

RORγt-expressing Tregs form a specialized subset of intestinal CD4+ Foxp3+ cells which is essential to maintain gut homeostasis and tolerance to commensal microbiota. Recently, c-Maf emerged as a critical factor in the regulation of RORγt expression in Tregs. However, aside from c-Maf signaling, the signaling pathways involved in the differentiation of RORγt+ Tregs and their possible interplay with c-Maf in this process are largely unknown. We show that RORγt+ Treg development is controled by positive as well as negative signals. Along with c-Maf signaling, signals derived from a complex microbiota, as well as IL-6/STAT3- and TGF-ß-derived signals act in favor of RORγt+ Treg development. Ectopic expression of c-Maf did not rescue RORγt expression in STAT3-deficient Tregs, indicating the presence of additional effectors downstream of STAT3. Moreover, we show that an inflammatory IFN-γ/STAT1 signaling pathway acts as a negative regulator of RORγt+ Treg differentiation in a c-Maf independent fashion. These data thus argue for a complex integrative signaling network that finely tunes RORγt expression in Tregs. The finding that type 1 inflammation impedes RORγt+ Treg development even in the presence of an active IL-6/STAT3 pathway further suggests a dominant negative effect of STAT1 over STAT3 in this process.

Dual effect of hemin on renal ischemia-reperfusion injury.

Acute kidney injury (AKI) is a major public health concern, which is contributing to serious hospital complications, chronic kidney disease (CKD) and even death. Renal ischemia-reperfusion injury (IRI) remains a leading cause of AKI. The stress-responsive enzyme, heme oxygenase-1 (HO-1) mediates protection against renal IRI and may be preventively induced using hemin prior to renal insult. This HO-1 induction pathway called hemin preconditioning is largely known to be effective. Therefore, HO-1 might be an interesting therapeutic target in case of predictable AKI (e.g. partial nephrectomy or renal transplantation). However, the use of hemin to mitigate established AKI remains poorly characterized. Mice underwent bilateral renal IRI for 26 min or sham surgery. After surgical procedure, animals were injected either with hemin (5 mg/kg) or vehicle. Twenty-four hours later, mice were sacrificed. Despite strong HO-1 induction, hemin-treated mice exhibited significant renal damage and oxidative stress as compared to vehicle-treated mice. Interestingly, higher dose of hemin is associated with more severe IRI-induced AKI in a dose-dependent relation. To determine whether hemin preconditioning remains efficient to dampen postoperative hemin-amplified IRI-induced AKI, we pretreated mice either with hemin (5 mg/kg) or vehicle 24 h prior to surgical procedure. Then, all mice (hemin- and vehicle-pretreated) received postoperative injection of hemin (5 mg/kg) to amplify IRI-induced AKI. In comparison to vehicle, prior administration of hemin to renal IRI mitigated hemin-amplified IRI-induced AKI as attested by fewer renal damage, inflammation and oxidative stress. In conclusion, hemin may have a dual effect on renal IRI, protective or deleterious, depending on the timing of its administration.

Activation of the endoplasmic reticulum stress sensor IRE1α by the vaccine adjuvant AS03 contributes to its immunostimulatory properties.

The oil-in-water emulsion Adjuvant System 03 (AS03) is one of the few adjuvants used in licensed vaccines. Previous work indicates that AS03 induces a local and transient inflammatory response that contributes to its adjuvant effect. However, the molecular mechanisms involved in its immunostimulatory properties are ill-defined. Upon intramuscular injection in mice, AS03 elicited a rapid and transient downregulation of lipid metabolism-related genes in the draining lymph node. In vitro, these modifications were associated with profound changes in lipid composition, alteration of endoplasmic reticulum (ER) morphology and activation of the unfolded protein response pathway. In vivo, treatment with a chemical chaperone or deletion of the ER stress sensor kinase IRE1α in myeloid cells decreased AS03-induced cytokine production and its capacity to elicit high affinity antigen-specific antibodies. In summary, our results indicate that IRE1α is a sensor for the metabolic changes induced by AS03 in monocytic cells and may constitute a canonical pathway that could be exploited for the design of novel vaccine adjuvants.

A Flow Cytometry-Based Protocol to Measure Lymphocyte Viability Upon Metabolic Stress.

Distinct lymphocyte subpopulations display discrete metabolic profiles and are differently affected by metabolic resource variations, making the analysis of lymphocyte survival in a complex tissue in response to metabolic stress highly challenging. Here we describe a flow cytometry-based method allowing simultaneous cell identification and viable cell counting in mixed lymphocyte populations without extensive cell subset purification procedures. The example provided herein illustrates the role of AMPK in T lymphocyte survival in response to the mitochondrial poison oligomycin.

Regulatory T cells constrain the TCR repertoire of antigen-stimulated conventional CD4 T cells.

To analyze the potential role of Tregs in controlling the TCR repertoire breadth to a non-self-antigen, a TCRß transgenic mouse model (EF4.1) expressing a limited, yet polyclonal naïve T-cell repertoire was used. The response of EF4.1 mice to an I-Ab-associated epitope of the F-MuLV envelope protein is dominated by clones expressing a Vα2 gene segment, thus allowing a comprehensive analysis of the TCRα repertoire in a relatively large cohort of mice. Control and Treg-depleted EF4.1 mice were immunized, and the extent of the Vα2-bearing, antigen-specific TCR repertoire was characterized by high-throughput sequencing and spectratyping analysis. In addition to increased clonal expansion and acquisition of effector functions, Treg depletion led to the expression of a more diverse TCR repertoire comprising several private clonotypes rarely observed in control mice or in the pre-immune repertoire. Injection of anti-CD86 antibodies in vivo led to a strong reduction in TCR diversity, suggesting that Tregs may influence TCR repertoire diversity by modulating costimulatory molecule availability. Collectively, these studies illustrate an additional mechanism whereby Tregs control the immune response to non-self-antigens.

Cyclophosphamide treatment regulates the balance of functional/exhausted tumor-specific CD8+ T cells.

An important question is how chemotherapy may (re-)activate tumor-specific immunity. In this study, we provide a phenotypic, functional and genomic analysis of tumor-specific CD8+ T cells in tumor (P815)-bearing mice, treated or not with cyclophosphamide. Our data show that chemotherapy favors the development of effector-type lymphocytes in tumor bed, characterized by higher KLRG-1 expression, lower PD-1 expression and increased cytotoxicity. This suggests re-engagement of T lymphocytes into the effector program. IFN-I appears involved in this remodeling. Our findings provide some insight into how cyclophosphamide regulates the amplitude and quality of tumor-specific immune responses.

The Transcription Factor c-Maf Promotes the Differentiation of Follicular Helper T Cells.

Follicular helper T cells (Tfh) have been identified as the primary cell subpopulation regulating B cell responses in germinal centers, thus supporting high-affinity antibody production. Among the transcription factors orchestrating Tfh cell differentiation and function, the role played by the proto-oncogene c-Maf remains poorly characterized. We report herein that selective loss of c-Maf expression in the T cell compartment results in defective development of Tfh cells in response to both antigen/adjuvant vaccinations and commensal intestinal bacteria. Accordingly, c-Maf expression in T cells was essential for the development and high-affinity antibody secretion in vaccinated animals. c-Maf was expressed early, concomitantly to BCL6, in Tfh cell precursors and found to regulate Tfh fate in a cell-autonomous fashion. Altogether, our findings reveal a novel, non-redundant, function for c-Maf in the differentiation of Tfh cells and the regulation of humoral immune responses to T-cell-dependent antigens.

Specific expression of heme oxygenase-1 by myeloid cells modulates renal ischemia-reperfusion injury.

Renal ischemia-reperfusion injury (IRI) is a major risk factor for delayed graft function in renal transplantation. Compelling evidence exists that the stress-responsive enzyme, heme oxygenase-1 (HO-1) mediates protection against IRI. However, the role of myeloid HO-1 during IRI remains poorly characterized. Mice with myeloid-restricted deletion of HO-1 (HO-1M-KO), littermate (LT), and wild-type (WT) mice were subjected to renal IRI or sham procedures and sacrificed after 24 hours or 7 days. In comparison to LT, HO-1M-KO exhibited significant renal histological damage, pro-inflammatory responses and oxidative stress 24 hours after reperfusion. HO-1M-KO mice also displayed impaired tubular repair and increased renal fibrosis 7 days after IRI. In WT mice, HO-1 induction with hemin specifically upregulated HO-1 within the CD11b+ F4/80lo subset of the renal myeloid cells. Prior administration of hemin to renal IRI was associated with significant increase of the renal HO-1+ CD11b+ F4/80lo myeloid cells in comparison to control mice. In contrast, this hemin-mediated protection was abolished in HO-1M-KO mice. In conclusion, myeloid HO-1 appears as a critical protective pathway against renal IRI and could be an interesting therapeutic target in renal transplantation.

Nicotinamide Phosphoribosyltransferase in Smooth Muscle Cells Maintains Genome Integrity, Resists Aortic Medial Degeneration, and Is Suppressed in Human Thoracic Aortic Aneurysm Disease.

RATIONALE: The thoracic aortic wall can degenerate over time with catastrophic consequences. Vascular smooth muscle cells (SMCs) can resist and repair artery damage, but their capacities decline with age and stress. Recently, cellular production of nicotinamide adenine dinucleotide (NAD+) via nicotinamide phosphoribosyltransferase (Nampt) has emerged as a mediator of cell vitality. However, a role for Nampt in aortic SMCs in vivo is unknown. OBJECTIVES: To determine whether a Nampt-NAD+ control system exists within the aortic media and is required for aortic health. METHODS AND RESULTS: Ascending aortas from patients with dilated aortopathy were immunostained for NAMPT, revealing an inverse relationship between SMC NAMPT content and aortic diameter. To determine whether a Nampt-NAD+ control system in SMCs impacts aortic integrity, mice with Nampt-deficient SMCs were generated. SMC-Nampt knockout mice were viable but with mildly dilated aortas that had a 43% reduction in NAD+ in the media. Infusion of angiotensin II led to aortic medial hemorrhage and dissection. SMCs were not apoptotic but displayed senescence associated-ß-galactosidase activity and upregulated p16, indicating premature senescence. Furthermore, there was evidence for oxidized DNA lesions, double-strand DNA strand breaks, and pronounced susceptibility to single-strand breakage. This was linked to suppressed poly(ADP-ribose) polymerase-1 activity and was reversible on resupplying NAD+ with nicotinamide riboside. Remarkably, we discovered unrepaired DNA strand breaks in SMCs within the human ascending aorta, which were specifically enriched in SMCs with low NAMPT. NAMPT promoter analysis revealed CpG hypermethylation within the dilated human thoracic aorta and in SMCs cultured from these tissues, which inversely correlated with NAMPT expression. CONCLUSIONS: The aortic media depends on an intrinsic NAD+ fueling system to protect against DNA damage and premature SMC senescence, with relevance to human thoracic aortopathy.