Macrophages integrate metabolic signals with immune activation, and their ability to handle fatty acids is central to preventing lipotoxicity and to sustaining effector functions. In cardiometabolic settings such as obesity, metabolic dysfunction-associated steatotic liver disease, and atherosclerosis, chronic exposure to excess free fatty acids and toxic lipid species, such as ceramides, disrupts organelle integrity, impairs efferocytosis, and skews macrophages toward pro-inflammatory phenotypes. This review examines how macrophages channel fatty acids into β-oxidation, glycerolipid synthesis and storage, sphingolipid production, and polyunsaturated fatty acid-derived lipid mediator biosynthesis to shape the balance among metabolic adaptation, inflammatory activation, resolution, and lipid-induced dysfunction. We also highlight lipin-1 as a regulatory node at a key branchpoint in macrophage lipid metabolism. By linking glycerolipid synthesis, lipid storage, mitochondrial metabolism, and inflammatory signaling, lipin-1 illustrates how lipid routing can influence macrophage function in cardiometabolic disease.

Macrophages are critical to maintaining and restoring tissue homeostasis during inflammation. The lipid metabolic state of macrophages influences their function and polarization, which is crucial to the resolution of inflammation. The contribution of lipid synthesis to proinflammatory macrophage responses is well understood. However, how lipid synthesis regulates proresolving macrophage responses needs to be better understood. Lipin-1 is a phosphatidic acid phosphatase with a transcriptional coregulatory activity that regulates lipid metabolism. We previously demonstrated that lipin-1 supports proresolving macrophage responses, and here, myeloid-associated lipin-1 is required for inflammation resolution, yet how lipin-1-regulated cellular mechanisms promote macrophage proresolution responses is unknown. We demonstrated that the loss of lipin-1 in macrophages led to increased free fatty acid, neutral lipid, and ceramide content and increased phosphorylation of acetyl-CoA carboxylase. The inhibition of the first step of lipid synthesis, the transport of citrate from the mitochondria, reduced lipid content and restored efferocytosis and inflammation resolution in lipin-1mKO mice and macrophages. Our findings suggest macrophage-associated lipin-1 restrains lipid synthesis, promoting proresolving macrophage function in response to proresolving stimuli.

Chronic inflammation drives the pathophysiology of many cardiometabolic diseases. Pro-resolving macrophages (Møs) resolve inflammation and restore tissue homeostasis via lipid metabolism-dependent efferocytosis. Understanding the regulatory pathways that control Mø lipid metabolic profiles is essential for uncovering mechanisms of inflammation resolution. We have shown that lipin-1, a phosphohydrolase and a transcriptional co-regulator, promotes inflammation resolution by enhancing β-oxidation and efferocytosis. Our current study aims to define the mechanisms by which Mø lipin-1 promotes β-oxidation, efferocytosis and inflammation resolution. Using myeloid-specific lipin-1 knockout (lipin-1mKO) mice and littermate controls, we show that lipin-1 facilitates mitochondrial fission, producing fragmented mitochondria with enhanced β-oxidation capacity compared to elongated mitochondria. Additionally, lentiviral transduction of lipin-1mKO Møs with specific truncated forms of lipin-1 suggests that a novel non-canonical activity of lipin-1 is sufficient to restore and augment efferocytosis. Our findings uncover a novel role for lipin-1 in coordinating mitochondrial structure and metabolic function in macrophages. This deeper understanding of lipin-1’s influence on mitochondrial dynamics and macrophage function advances our knowledge of the cellular mechanisms underlying inflammation resolution and may inform future investigations into metabolic regulation in immune responses. Supported by NIH under grant number P20GM134974 and R01HL163106; and CCDS under grant number CCDS000013 Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

This review examines anti-atherogenic mechanisms and the crucial role of efferocytosis in promoting inflammation resolution, with a focus on innovative, resolution-based therapeutic strategies that aim to restore vascular homeostasis and mitigate atherosclerosis progression. Atherosclerosis, a chronic inflammatory condition, is exacerbated by impaired efferocytosis, which contributes to plaque instability and the expansion of the necrotic core. Advanced molecular and cellular profiling has revealed diverse macrophage populations and their metabolic adaptations during efferocytosis, which drive the production of resolving mediators essential for tissue repair. Dysregulated signaling and metabolic pathways disrupt the efficient clearance of apoptotic cells, exacerbating inflammation. Molecular regulators, such as microRNAs, further impact efferocytosis, governing cardiovascular outcomes. Resolution-based therapies, including specialized pro-resolving mediators, peptides, and metabolites, enhance the successive clearance of apoptotic cells while maintaining host immune function, offering advantages over traditional immunosuppressive approaches. Additionally, vaccines targeting disease-specific antigens show promise in eliciting protective immune responses that can help ameliorate atherosclerosis. Efferocytosis is a key regulator of inflammation resolution in atherosclerosis, linking macrophage metabolism to plaque stability. Its disruption drives disease progression, but emerging therapies targeting resolution pathways, metabolic reprogramming, and immune modulation hold the potential for effective interventions. Advances in profiling technologies and targeted delivery systems will address translational challenges, paving the way for precision medicine in treating atherosclerotic cardiovascular disease.

Altered hemodynamics is a key factor for atherosclerosis. For decades, endothelial cell (EC) responses to fluid-generated wall shear stress have been the central focus for atherogenesis. However, circulating blood is not a cell-free fluid, it contains mechanosensitive red blood cells (RBCs) that are also subjected to altered hemodynamics and release a large amount of ATP, but their impact on atherosclerosis has been overlooked. The focus of this study is the role of shear stress (SS)-induced RBC-released ATP in atherosclerosis. Hypercholesterolemic mouse models with and without RBC-Pannexin 1 deletion were used for the study. Results showed that SS-induced release of ATP from RBCs was at μM concentrations, three-orders of magnitude higher than that from other cell types. Suppression of RBC-released ATP via deletion of Pannexin 1, a mechanosensitive ATP-permeable channel, reduced high fat diet-induced aortic plaque burden by 40-60%. Importantly, the location and the extent of aortic atherosclerotic lesions spatially matched with the ATP deposition profile at aortic wall predicted by a computational fluid dynamic (CFD) model. Furthermore, hypercholesterolemia increases EC susceptibility to ATP with potentiated increase in [Ca2+]i, an initial signaling for aortic EC barrier dysfunction, and an essential cause for lipid accumulation and inflammatory cell infiltration. The computational prediction also provides a physics-based explanation for RBC-released ATP-induced sex disparities in atherosclerosis. Our study reveals an important role of RBC-released ATP in the initiation and progression of atherosclerosis. These novel findings provide a more comprehensive view of how altered hemodynamics and systemic risk factors synergistically contribute to atherosclerosis.

The Center for Cardiovascular Diseases and Sciences at LSU Health Sciences Center at Shreveport supports cardiovascular disease research at multiple levels from predoctoral fellows to established faculty. To extend our support to undergraduate students, we developed the Cardiovascular Undergraduate Research Initiative fOr Underrepresented Students (CURIOUS). CURIOUS is an 8-week summer research program (with housing) funded by an NHLBI R25 grant, and is targeted towards underrepresented groups interested in pursuing cardiovascular research as part of their scientific/clinical career goals. We primarily recruit in our region, but the program is open to students across the USA. CURIOUS provides an intensive experience at the bench alongside multiple enrichments. Professional development includes meeting administrators involved in admissions, and students from our schools of Graduate Studies, Medicine and Allied Health Professions, financial advice for planning graduate education, CV writing, and presentation skills. The students also have discussions with faculty about challenges & opportunities for underrepresented minorities in research, and diversity, inclusion & equity. Other enrichments cover cardiovascular basics and state-of-the-art techniques available on our campus. Students receive training in the responsible conduct of research, held in conjunction with other undergraduate programs and graduate students. We invite a nationally recognized cardiovascular researcher to give a seminar and meet with the CURIOUS students. The program ends with poster presentations for all first-year students. Several students are selected to present at national/international conferences with their mentor. In addition, 1-2 students are invited back for a second year of the program during which they have more time for research, advanced enrichments, and an oral presentation at the end of the program. To date, the acceptance rate has been 26%, with 39 students having completed the 1st year and 2 returning students. CURIOUS provided 59% of students with their first research experience. 80% of students gave the program the top rating of excellent. Based on end-of-program feedback, we have improved pre-program interaction with mentors, refined enrichments, streamlined the on-boarding process, and enhanced social activities. Of the 25 students who have graduated college, 2 are in MD/PhD programs, 1 is in a PhD program, and 10 are in medical school, with 11 others planning one of these programs (10 are working in science-related jobs during gap years). Overall, the program has successfully improved the students’ consideration of including cardiovascular research as part of their future career. Funded by the NIH National Heart, Lung and Blood Institute, Award R25HL147665, to KYS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heart disease is the leading cause of death in the United States and atherosclerosis accounts for nearly 75% of all deaths from heart disease. Atherosclerosis is a chronic inflammatory disease and mitochondrial reactive oxygen species (mtROS) are critical contributors to disease development. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that maintains mitochondrial NADPH pools and supports the enzymatic degradation of mtROS. Preliminary data indicates that NNT expression is decreased in the endothelium of human atherosclerotic plaques and we sought to test the hypothesis that the loss of NNT increases mtROS and promotes atherosclerosis by enhancing endothelial and vascular dysfunction. Previous we have demonstrated that the loss of NNT was associated with increased vascular ROS production, plaque formation, and plaque size in response to treatment with PCSK9 and high fat diet (HFD). We now show that the loss of NNT in endothelial specific NNT knockout mice promotes enhanced endothelial VCAM-1 expression in response to HFD and disturbed flow. Similarly, in vitro, the loss of NNT promoted both VCAM-1 and ICAM-1 expression in response to oxLDL. Increased adhesion molecule expression associated with the loss of NNT also augmented monocyte adhesion in response to oxLDL consistent with vascular inflammation observed in early atherosclerosis. Additionally, macrophages isolated from NNT knockout mice display enhanced M1 polarization, decreased lipid uptake, and impaired lipid utilization. Co- treatment with the mitochondria targeted antioxidant MitoEbselen is able to normalize both endothelial and macrophage function, underscoring a critical role for mtROS in promoting an inflammatory phenotype in these cells. In both global and endothelial cell specific NNT knockout mice the loss of NNT was associated with increased necrotic core formation in response to PCSK9 treatment and 16 weeks of high fat diet. Taken together, these data indicate that decreased NNT expression promotes endothelial activation, increased inflammatory cell trafficking, macrophage metabolic dysfunction and M1 polarization that could contribute to an acceleration of necrotic core development and severe atherosclerotic disease. NIH P20GM121307. NIH 1 R01 HL145171-01A1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glucocorticoids acting via the glucocorticoid receptors (GR) are key regulators of metabolism and the stress response. However, uncontrolled or excessive GR signaling adversely affects adipose tissue, including endocrine, immune, and metabolic functions. Inflammation of the adipose tissue promotes systemic metabolic dysfunction; however, the molecular mechanisms underlying the role of adipocyte GR in regulating genes associated with adipose tissue inflammation are poorly understood. We performed in vivo studies using adipocyte-specific GR knockout mice in conjunction with in vitro studies to understand the contribution of adipocyte GR in regulating adipose tissue immune homeostasis. Our findings show that adipocyte-specific GR signaling regulates adipokines at both mRNA and plasma levels and immune regulatory (Coch, Pdcd1, Cemip, and Cxcr2) mRNA gene expression, which affects myeloid immune cell presence in white adipose tissue. We found that, in adipocytes, GR directly influences Cxcr2. This chemokine receptor promotes immune cell migration, indirectly affecting Pdcd1 and Cemip gene expression in nonadipocyte or stromal cells. Our findings suggest that GR adipocyte signaling suppresses inflammatory signals, maintaining immune homeostasis. We also found that GR signaling in adipose tissue in response to stress is sexually dimorphic. Understanding the molecular relationship between GR signaling and adipose tissue inflammation could help develop potential targets to improve local and systemic inflammation, insulin sensitivity, and metabolic health.

Bordetella spp. are respiratory pathogens equipped with immune evasion mechanisms. We previously characterized a Bordetella bronchiseptica mutant (RB50ΔbtrS) that fails to suppress host responses, leading to rapid clearance and long-lasting immunity against reinfection. This work revealed eosinophils as an exclusive requirement for RB50ΔbtrS clearance. We also show that RB50ΔbtrS promotes eosinophil-mediated B/T cell recruitment and inducible bronchus-associated lymphoid tissue (iBALT) formation, with eosinophils being present throughout iBALT for Th17 and immunoglobulin A (IgA) responses. Finally, we provide evidence that XCL1 is critical for iBALT formation but not maintenance, proposing a novel role for eosinophils as facilitators of adaptive immunity against B. bronchiseptica. RB50ΔbtrS being incapable of suppressing eosinophil effector functions illuminates active, bacterial targeting of eosinophils to achieve successful persistence and reinfection. Overall, our discoveries contribute to understanding cellular mechanisms for use in future vaccines and therapies against Bordetella spp. and extension to other mucosal pathogens.

Rationale:SARS-CoV-2 entry into host cells is facilitated by endogenous and exogenous proteases that proteolytically activate the spike glycoprotein and antiproteases inhibiting this process. Understanding the key actors in viral entry is crucial for advancing knowledge of virus tropism, pathogenesis, and potential therapeutic targets. Objectives:We aimed to investigate the role of naïve serum and alpha-1-antitrypsin (AAT) in inhibiting protease-mediated SARS-CoV-2 entry and explore the implications of AAT deficiency on susceptibility to different SARS-CoV-2 variants. Findings:Our study demonstrates that naïve serum exhibits significant inhibition of SARS-CoV-2 entry, with AAT identified as the major serum protease inhibitor potently restricting entry. Using pseudoparticles, replication-competent pseudoviruses, and authentic SARS-CoV-2, we show that AAT inhibition occurs at low concentrations compared with those in serum and bronchoalveolar tissues, suggesting physiological relevance. Furthermore, sera from subjects with an AAT-deficient genotype show reduced ability to inhibit entry of both Wuhan-Hu-1 (WT) and B.1.617.2 (Delta) but exhibit no difference in inhibiting B.1.1.529 (Omicron) entry. Conclusions:AAT may have a variant-dependent therapeutic potential against SARS-CoV-2. Our findings highlight the importance of further investigating the complex interplay between proteases, antiproteases, and spike glycoprotein activation in SARS-CoV-2 and other respiratory viruses to identify potential therapeutic targets and improve understanding of disease pathogenesis.

Sickle Cell Disease (SCD) is an inherited hemoglobinopathy characterized by a pro-inflammatory, pro-thrombotic phenotype. Platelet-leukocyte aggregates (PLAs), a hallmark of thrombo-inflammatory diseases, are elevated in SCD. While neutrophils play a role in aggregate formation, little is known about other leukocyte subpopulations. Additionally, SCD red blood cells (RBCs) can become adherent but evidence showing how they participate in aggregate formation is limited. The first aim of this study was to develop a flow cytometry protocol to test our hypothesis that red blood cell aggregate formation with different leukocyte subpopulations and/or platelets is elevated in SCD. The second aim of this study was to compare the PLA findings from our new no-lysis protocol with those from a standard lysis protocol.Systemic blood was collected from the tail vein of C57Bl/6J (WT) and Townes (SCD) mice (n=6-10/grp). Flow cytometry analysis was performed with lysed and non-lysed blood. PLAs were identified by double-positive staining for CD45+ (leukocytes) and CD41+ (platelets). Leukocyte subpopulations were identified as Ly6G+ (neutrophils-PNAs), CD115+ (monocytes-PMAs), or Ly6G-/CD115- (primarily lymphocytes-PLyAs). RBC aggregates were further identified as Ter119+. Student’s t-test was used for statistical analysis with significance set at p<0.05.Total platelet and leukocyte counts (including individual neutrophil, monocyte, and lymphocyte subpopulations) were significantly increased in SCD mice. Total PLAs were significantly higher in SCD versus WT mice with the increase being driven by the significant increases in PNAs and PMAs in the lysis protocol whereas in the no-lysis protocol it was driven by significant increases in PNAs and PLyAs. Overall numbers of circulating PLAs were about 5-fold higher for both WT and SCD mice in the lysis protocol compared to the no-lysis protocol. RBC-leukocyte-aggregates (RLAs), RBC-platelet-aggregates (RPAs), and RBC-platelet-leukocyte-aggregates (RPLAs) were all significantly increased more than two-fold in SCD mice compared to WT mice.Our findings of increased circulating PLAs and PNAs in SCD mice reflect what is seen in human SCD. The elevated numbers of PLAs in both WT and SCD mice from the lysis protocol compared with the no-lysis protocol suggests that the additional lysis and processing steps introduces an artifact of excessive aggregation, with the no-lysis protocol potentially being more representative. Furthermore, using our new no-lysis protocol we were able to detect increased numbers of all aggregates involving RBCs in SCD mice. We have characterized a model to further investigate the mechanisms underlying the systemic thrombo-inflammatory phenotype of SCD, and better understand RBC interactions with leukocyte subpopulations, platelets, and PLAs. Funded by T32HL155022 from the NIH NHLBI, awarded to A. Wayne Orr and Karen Y. Stokes, Center for Cardiovascular Diseases and Sciences, LSU Health Sciences Center Shreveport, and by 5R01HL134959 from the NIH NHLBI awarded to Karen Y. Stokes. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Free fatty acid accumulation in macrophages alters cellular metabolism, leading to failed inflammation resolution that contributes to cardiometabolic pathologies such as atherosclerotic cardiovascular disease. Free fatty acids are either broken down by β-oxidation, stored in glycerolipids, or incorporated into sphingolipids (e.g., ceramides). Ceramide synthesis inhibits several pro-resolving macrophage functions, such as β-oxidation and efferocytosis, needed for inflammation resolution. The regulatory signals that control free fatty acid incorporation into lipids (e.g., lipid channeling) for proper macrophage function are not well understood. Lipin-1 is a phosphatidic acid phosphatase with an independent transcriptional coregulatory activity that controls cellular lipid homeostasis. Using genetically engineered mice and bone marrow-derived macrophages, we investigated the contribution of lipin-1 on macrophage pro-resolving functions. Mice lacking myeloid-specific lipin-1 had defects in the clearance of apoptotic cells in a zymosan model of inflammation resolution, and these mice had increased atherosclerotic plaques and necrotic cores in a model of atherosclerosis and a delay excisional wound closure. Bone marrow-derived macrophages lacking lipin-1 showed a striking pattern of dysregulated lipid metabolism in which il-4 stimulation promoted ceramide synthesis over β-oxidation. Additionally, lipin-1 deficient macrophages had reduced phagocytosis of apoptotic cells. Our work provides evidence that lipin-1 promotes β-oxidation while inhibiting ceramide synthesis during free fatty acid accumulation in macrophages to allow for responses that promote inflammation resolution. Supported by grants from NIH (1 R01HL131844-04) and NIH (1P20GM134974-01A1)

Atherosclerosis is a chronic inflammatory disease effecting large and medium sized arteries that leads to the buildup of plaque that contributes to myocardial infraction, stroke, and other pathologies. Mounting evidence has identified mitochondrial reactive oxygen species (ROS) as a key contributor to plaque formation and progression. A inner mitochondrial membrane enzyme Nicotinamide nucleotide transhydrogenase (NNT) sustains NADPH pools required for the mitochondrial antioxidant systems. We have observed that the expression of NNT is decreased in severe human atherosclerotic plaques leading us to hypothesize that the loss of NNT contributes to increased mitochondrial ROS and drives the progression of atherosclerosis by enhancing endothelial and vascular dysfunction. Utilizing AAV/PCSK9 and HFD induced models of early and late-stage atherosclerosis, we found that NNT knockout (NNT KO) mice display significantly increased VCAM-1 expression in aortic endothelial cells during early atherosclerosis and significantly larger necrotic cores in late-stage atherosclerosis. Additionally, NNT KO mice displayed a significantly thinner fibrous cap in late-stage plaques. Similar results were observed in vitro where the loss of NNT in human aortic endothelial cells exacerbated oxLDL induced VCAM-1 expression that was associated with decreased mitochondrial NADPH levels, glutathione peroxidase (Gpx2) activity, and increased H 2 O 2 production. The observed increase in necrotic core area in NNT KO mice is suggestive of increased inflammatory cell recruitment to the plaque. Furthermore, bone marrow derived macrophages (BMDM) from NNT KO mice display decreased lipid content, poor lipid uptake and a predisposition to differentiate into a proinflammatory M1 phenotype. Based on these studies, we conclude that the loss of NNT contributes to unstable plaques with large necrotic cores, mitochondrial ROS driven endothelial dysfunction, increased inflammatory cell recruitment, and enhanced M1 macrophage activity. The novel relationship between vascular NNT expression and plaque severity in human samples implicates NNT as a novel clinical target in atherosclerosis.

Macrophages are critical to maintaining and restoring tissue homeostasis during inflammation. The lipid metabolic state of macrophages influences their function, but a deeper understanding of how lipid metabolism is regulated in pro-resolving macrophage responses is needed. Lipin-1 is a phosphatidic acid phosphatase with a transcriptional coregulatory activity (TC) that regulates lipid metabolism. We previously demonstrated that lipin-1 supports pro-resolving macrophage responses, and here, myeloid-associated lipin-1 is required for inflammation resolution, yet how lipin-1-regulated cellular mechanisms promote macrophage pro-resolution responses is unknown. We demonstrated that the loss of lipin-1 in macrophages led to increased free fatty acid, neutral lipid, and ceramide content and increased phosphorylation of acetyl-CoA carboxylase. The inhibition of the first step of lipid synthesis and transport of citrate from the mitochondria in macrophages reduced lipid content and restored efferocytosis and inflammation resolution in lipin-1 m KO macrophages and mice. Our findings suggest macrophage-associated lipin-1 restrains lipid synthesis, promoting pro-resolving macrophage function in response to pro-resolving stimuli. Teaser Lipin 1 blockade of lipid biosynthesis inducing mitochondrial citrate export promotes efferocytosis and inflammation resolution.

ABSTRACTA characteristic that differentiates pathogenic and opportunistic bacteria is that pathogens have been selected by their ability to suppress host inflammatory responses allowing colonization and persistence. Bordetella spp. are respiratory pathogens characterized for the arsenal of mechanisms they use to manipulate host immune responses. We have previously characterized a B. bronchiseptica mutant, RB50ΔbtrS, that is not able to suppress host immune responses, resulting not only in rapid clearance of the infection but also long-term lung sterilizing immunity against reinfection with the three classical Bordetella spp. Interestingly, this strong immune response requires eosinophils. In this work our results indicate that wildtype B. bronchiseptica, RB50, blocks eosinophil pro-inflammatory functions to prevent the rapid recruitment of B and T cells to the lung that results in iBALT formation. Moreover, eosinophils promote a TH17 microenvironment within the iBALT that might be responsible for the long-term robust protective immunity generated by infection with this mutant. Overall, this work provides a novel role for eosinophils as promoters of adaptive immune responses and protective immunity, while also indicating that bacteria actively manipulate those cells to promote long-term persistence and reinfection.

Heart disease is the leading cause of death in the United States and atherosclerosis accounts for nearly 75% of all deaths from heart disease. Atherosclerosis is a chronic inflammatory disease and mitochondrial reactive oxygen species (mtROS) are critical contributors to disease development. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that maintains mitochondrial NADPH pools and supports the enzymatic degradation of mtROS. Preliminary data indicates that NNT expression is decreased in the endothelium of human atherosclerotic plaques and we sought to test the hypothesis that the loss of NNT increases mtROS and promotes atherosclerosis by enhancing endothelial and vascular dysfunction. Previous we have demonstrated that the loss of NNT was associated with increased vascular ROS production, plaque formation, and plaque size in response to treatment with PCSK9 and high fat diet (HFD). We now show that the loss of NNT in endothelial specific NNT knockout mice promotes enhanced endothelial VCAM-1 expression in response to HFD and disturbed flow. Similarly, in vitro , the loss of NNT promoted both VCAM-1 and ICAM-1 expression in response to oxLDL. Increased adhesion molecule expression associated with the loss of NNT also augmented monocyte adhesion in response to oxLDL consistent with vascular inflammation observed in early atherosclerosis. Additionally, macrophages isolated from NNT knockout mice display enhanced M1 polarization, decreased lipid uptake, and impaired lipid utilization. Co-treatment with the mitochondria targeted antioxidant MitoEbselen is able to normalize both endothelial and macrophage function, underscoring a critical role for mtROS in promoting an inflammatory phenotype in these cells. In both global and endothelial cell specific NNT knockout mice the loss of NNT was associated with increased necrotic core formation in response to PCSK9 treatment and 16 weeks of high fat diet. Taken together, these data indicate that decreased NNT expression promotes endothelial activation, increased inflammatory cell trafficking, macrophage metabolic dysfunction and M1 polarization that could contribute to an acceleration of necrotic core development and severe atherosclerotic disease.