Methamphetamine Use and Cardiovascular Disease.

While the opioid epidemic has garnered significant attention, the use of methamphetamines is growing worldwide independent of wealth or region. Following overdose and accidents, the leading cause of death in methamphetamine users is cardiovascular disease, because of significant effects of methamphetamine on vasoconstriction, pulmonary hypertension, atherosclerotic plaque formation, cardiac arrhythmias, and cardiomyopathy. In this review, we examine the current literature on methamphetamine-induced changes in cardiovascular health, discuss the potential mechanisms regulating these varied effects, and highlight our deficiencies in understanding how to treat methamphetamine-associated cardiovascular dysfunction.

Macrophage-Associated Lipin-1 Enzymatic Activity Contributes to Modified Low-Density Lipoprotein-Induced Proinflammatory Signaling and Atherosclerosis.

OBJECTIVE: Macrophage proinflammatory responses induced by modified low-density lipoproteins (modLDL) contribute to atherosclerotic progression. How modLDL causes macrophages to become proinflammatory is still enigmatic. Macrophage foam cell formation induced by modLDL requires glycerolipid synthesis. Lipin-1, a key enzyme in the glycerolipid synthesis pathway, contributes to modLDL-elicited macrophage proinflammatory responses in vitro. The objective of this study was to determine whether macrophage-associated lipin-1 contributes to atherogenesis and to assess its role in modLDL-mediated signaling in macrophages. APPROACH AND RESULTS: We developed mice lacking lipin-1 in myeloid-derived cells and used adeno-associated viral vector 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (adeno-associated viral vector 8-proprotein convertase subtilisin/kexin type 9) to induce hypercholesterolemia and plaque formation. Mice lacking myeloid-associated lipin-1 had reduced atherosclerotic burden compared with control mice despite similar plasma lipid levels. Stimulation of bone marrow-derived macrophages with modLDL activated a persistent protein kinase Cα/ßII-extracellular receptor kinase1/2-jun proto-oncogene signaling cascade that contributed to macrophage proinflammatory responses that was dependent on lipin-1 enzymatic activity. CONCLUSIONS: Our data demonstrate that macrophage-associated lipin-1 is atherogenic, likely through persistent activation of a protein kinase Cα/ßII-extracellular receptor kinase1/2-jun proto-oncogene signaling cascade that contributes to foam cell proinflammatory responses. Taken together, these results suggest that modLDL-induced foam cell formation and modLDL-induced macrophage proinflammatory responses are not independent consequences of modLDL stimulation but rather are both directly influenced by enhanced lipid synthesis.

EphA2 Expression Regulates Inflammation and Fibroproliferative Remodeling in Atherosclerosis.

BACKGROUND: Atherosclerotic plaque formation results from chronic inflammation and fibroproliferative remodeling in the vascular wall. We previously demonstrated that both human and mouse atherosclerotic plaques show elevated expression of EphA2, a guidance molecule involved in cell-cell interactions and tumorigenesis. METHODS: Here, we assessed the role of EphA2 in atherosclerosis by deleting EphA2 in a mouse model of atherosclerosis (Apoe-/-) and by assessing EphA2 function in multiple vascular cell culture models. After 8 to 16 weeks on a Western diet, male and female mice were assessed for atherosclerotic burden in the large vessels, and plasma lipid levels were analyzed. RESULTS: Despite enhanced weight gain and plasma lipid levels compared with Apoe-/- controls, EphA2-/-Apoe-/- knockout mice show diminished atherosclerotic plaque formation, characterized by reduced proinflammatory gene expression and plaque macrophage content. Although plaque macrophages express EphA2, EphA2 deletion does not affect macrophage phenotype, inflammatory responses, and lipid uptake, and bone marrow chimeras suggest that hematopoietic EphA2 deletion does not affect plaque formation. In contrast, endothelial EphA2 knockdown significantly reduces monocyte firm adhesion under flow. In addition, EphA2-/-Apoe-/- mice show reduced progression to advanced atherosclerotic plaques with diminished smooth muscle and collagen content. Consistent with this phenotype, EphA2 shows enhanced expression after smooth muscle transition to a synthetic phenotype, and EphA2 depletion reduces smooth muscle proliferation, mitogenic signaling, and extracellular matrix deposition both in atherosclerotic plaques and in vascular smooth muscle cells in culture. CONCLUSIONS: Together, these data identify a novel role for EphA2 in atherosclerosis, regulating both plaque inflammation and progression to advanced atherosclerotic lesions. Cell culture studies suggest that endothelial EphA2 contributes to atherosclerotic inflammation by promoting monocyte firm adhesion, whereas smooth muscle EphA2 expression may regulate the progression to advanced atherosclerosis by regulating smooth muscle proliferation and extracellular matrix deposition.

Differential arterial and venous endothelial redox responses to oxidative stress.

OBJECTIVE: Oxidative stress is a central event linked with endothelial dysfunction and inflammation in several vascular pathologies, marked by over-production of ROS and concomitant decreases in antioxidants, for example GSH. Here, we distinguish endothelial oxidative stress regulation and associated functional disparities in the two main vascular conduits, (arteries and veins) following decreases in GSH. METHODS: MAECs and VCECs were used as models of arterial and venular endothelium, respectively, and BSO (0-100 µmol/L) was used to indirectly increase cellular oxidative stress. Inflammatory responses were measured using immune cell attachment and immunoblotting for endothelial cell adhesion molecule (ICAM-1, VCAM-1) expression, altered cell proliferation, and wound healing. RESULTS: MAECs and VCECs exhibited differential responses to oxidative stress produced by GSH depletion with VCECs exhibiting greater sensitivity to oxidative stress. Compared to MAECs, VCECs showed a significantly increased inflammatory profile and a decreased proliferative phenotype in response to decreases in GSH levels. CONCLUSIONS: Arterial and venous endothelial cells exhibit differential responses to oxidant stress, and decreases in GSH:GSSG are more exacerbated in venous endothelial cells. Specific pathogenesis in these vascular conduits, with respect to oxidant stress handling, warrants further study, especially considering surgical interventions such as Coronary artery bypass grafting that use both interchangeably.

Absence of Nicotinamide Nucleotide Transhydrogenase in C57BL/6J Mice Exacerbates Experimental Atherosclerosis.

BACKGROUND: Mitochondrial reactive oxygen species (ROS) contribute to inflammation and vascular remodeling during atherosclerotic plaque formation. C57BL/6N (6N) and C57BL/6J (6J) mice display distinct mitochondrial redox balance due to the absence of nicotinamide nucleotide transhydrogenase (NNT) in 6J mice. We hypothesize that differential NNT expression between these animals alters plaque development. METHODS: 6N and 6J mice were treated with AAV8-PCSK9 (adeno-associated virus serotype 8/proprotein convertase subtilisin/kexin type 9) virus leading to hypercholesterolemia, increased low-density lipoprotein, and atherosclerosis in mice fed a high-fat diet (HFD). Mice were co-treated with the mitochondria-targeted superoxide dismutase mimetic MitoTEMPO to assess the contribution of mitochondrial ROS to atherosclerosis. RESULTS: Baseline and HFD-induced vascular superoxide is increased in 6J compared to 6N mice. MitoTEMPO diminished superoxide in both groups demonstrating differential production of mitochondrial ROS among these strains. PCSK9 treatment and HFD led to similar increases in plasma lipids in both 6N and 6J mice. However, 6J animals displayed significantly higher levels of plaque formation. MitoTEMPO reduced plasma lipids but did not affect plaque formation in 6N mice. In contrast, MitoTEMPO surprisingly increased plaque formation in 6J mice. CONCLUSION: These data indicate that loss of NNT increases vascular ROS production and exacerbates atherosclerotic plaque development.

The arterial microenvironment: the where and why of atherosclerosis.

The formation of atherosclerotic plaques in the large and medium sized arteries is classically driven by systemic factors, such as elevated cholesterol and blood pressure. However, work over the past several decades has established that atherosclerotic plaque development involves a complex coordination of both systemic and local cues that ultimately determine where plaques form and how plaques progress. Although current therapeutics for atherosclerotic cardiovascular disease primarily target the systemic risk factors, a large array of studies suggest that the local microenvironment, including arterial mechanics, matrix remodelling and lipid deposition, plays a vital role in regulating the local susceptibility to plaque development through the regulation of vascular cell function. Additionally, these microenvironmental stimuli are capable of tuning other aspects of the microenvironment through collective adaptation. In this review, we will discuss the components of the arterial microenvironment, how these components cross-talk to shape the local microenvironment, and the effect of microenvironmental stimuli on vascular cell function during atherosclerotic plaque formation.

Janus kinase 3 activity is necessary for phosphorylation of cytosolic phospholipase A2 and prostaglandin E2 synthesis by macrophages infected with Francisella tularensis live vaccine strain.

Francisella tularensis, the causative agent of tularemia, modulates the host immune response to gain a survival advantage within the host. One mechanism of immune evasion is the ability of F. tularensis to induce the synthesis of the small lipid mediator prostaglandin E2 (PGE2), which alters the host T cell response making the host more susceptible to Francisella growth. PGE2 is synthesized by a tightly regulated biosynthetic pathway following stimulation. The synthesis of PGE2 begins with the liberation of arachidonic acid (AA) from membrane phospholipids by cytosolic phospholipase A2 (cPLA2). AA is subsequently converted to the unstable intermediate PGH2 by cyclooxygenase-2 (COX-2), and PGH2 undergoes an isomerization reaction to generate PGE2. Our objective was to identify F. tularensis-activated host signaling pathways that regulate the activity of the enzymes in the PGE2-biosynthetic pathway. In this study, we show that cPLA2, p38 mitogen-activated protein kinase (MAPK), and Janus kinase 3 (JAK3) signaling are necessary for F. tularensis-induced PGE2 production. Inhibition of JAK3 activity reduced the phosphorylation of cPLA2 and COX-2 protein levels. In addition, JAK3 regulates cPLA2 phosphorylation independent of transcription. Moreover, p38 MAPK activity is required for F. tularensis-induced COX-2 protein synthesis, but not for the phosphorylation of cPLA2. This research highlights a unique signaling axis in which JAK3 and p38 MAPK regulate the activity of multiple enzymes of the PGE2-biosynthetic pathway in macrophages infected with F. tularensis.

Francisella tularensis LVS induction of prostaglandin biosynthesis by infected macrophages requires specific host phospholipases and lipid phosphatases.

Francisella tularensis induces the synthesis of prostaglandin E(2) (PGE(2)) by infected macrophages to alter host immune responses, thus providing a survival advantage to the bacterium. We previously demonstrated that PGE(2) synthesis by F. tularensis-infected macrophages requires cytosolic phospholipase A2 (cPLA(2)), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES1). During inducible PGE(2) synthesis, cPLA(2) hydrolyzes arachidonic acid (AA) from cellular phospholipids to be converted to PGE(2). However, in F. tularensis-infected macrophages we observed a temporal disconnect between Ser505-cPLA(2) phosphorylation (a marker of activation) and PGE(2) synthesis. These results suggested to us that cPLA(2) is not responsible for the liberation of AA to be converted into PGE(2) by F. tularensis-infected macrophages. Utilizing small-molecule inhibitors, we demonstrated that phospholipase D and diacylglycerol lipase were required for providing AA for PGE(2) biosynthesis. cPLA(2), on the other hand, was required for macrophage cytokine responses to F. tularensis. We also demonstrated for the first time that lipin-1 and PAP2a contribute to macrophage inflammation in response to F. tularensis. Our results identify both an alternative pathway for inducible PGE(2) synthesis and a role for lipid-modifying enzymes in the regulation of macrophage inflammatory function.

Infection with Francisella tularensis LVS clpB leads to an altered yet protective immune response.

Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo. We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-γ) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB, can serve as a model for the rational design of attenuated vaccines.

Lipin-1 restrains macrophage lipid synthesis to promote inflammation resolution.

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-1mKO macrophages and mice. Our findings suggest macrophage-associated lipin-1 restrains lipid synthesis, promoting pro-resolving macrophage function in response to pro-resolving stimuli.

Adipocyte Glucocorticoid Receptor Inhibits Immune Regulatory Genes to Maintain Immune Cell Homeostasis in Adipose Tissue.

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.

Alpha-1-antitrypsin and its variant-dependent role in COVID-19 pathogenesis.

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.

Methamphetamine causes cardiovascular dysfunction via cystathionine gamma lyase and hydrogen sulfide depletion.

Methamphetamine (METH) is an addictive illicit drug used worldwide that causes significant damage to blood vessels resulting in cardiovascular dysfunction. Recent studies highlight increased prevalence of cardiovascular disease (CVD) and associated complications including hypertension, vasospasm, left ventricular hypertrophy, and coronary artery disease in younger populations due to METH use. Here we report that METH administration in a mouse model of 'binge and crash' decreases cardiovascular function via cystathionine gamma lyase (CSE), hydrogen sulfide (H2S), nitric oxide (NO) (CSE/H2S/NO) dependent pathway. METH significantly reduced H2S and NO bioavailability in plasma and skeletal muscle tissues co-incident with a significant reduction in flow-mediated vasodilation (FMD) and blood flow velocity revealing endothelial dysfunction. METH administration also reduced cardiac ejection fraction (EF) and fractional shortening (FS) associated with increased tissue and perivascular fibrosis. Importantly, METH treatment selectively decreased CSE expression and sulfide bioavailability along with reduced eNOS phosphorylation and NO levels. Exogenous sulfide therapy or endothelial CSE transgenic overexpression corrected cardiovascular and associated pathological responses due to METH implicating a central molecular regulatory pathway for tissue pathology. These findings reveal that therapeutic intervention targeting CSE/H2S bioavailability may be useful in attenuating METH mediated cardiovascular disease.

Identification of T-cell epitopes in Francisella tularensis using an ordered protein array of serological targets.

Francisella tularensis is a Gram-negative intracellular bacterium that is the causative agent of tularaemia. Concerns regarding its use as a bioterrorism agent have led to a renewed interest in the biology of infection, host response and pathogenesis. A robust T-cell response is critical to confer protection against F. tularensis. However, characterization of the cellular immune response has been hindered by the paucity of tools to examine the anti-Francisella immune response at the molecular level. We set out to combine recent advances of genomics with solid-phase antigen delivery coupled with a T-cell functional assay to identify T-cell epitopes. A subset of clones, encoding serological targets, was selected from an F. tularensis SchuS4 ordered genomic library and subcloned into a bacterial expression vector to test the feasibility of this approach. Proteins were expressed and purified individually employing the BioRobot 3000 in a semi-automated purification method. The purified proteins were coupled to beads, delivered to antigen-presenting cells for processing, and screened with Francisella-specific T-cell hybridomas of unknown specificity. We identified cellular reactivity against the pathogenicity protein IglB, and the chaperone proteins GroEL and DnaK. Further analyses using genetic deletions and synthetic peptides were performed to identify the minimal peptide epitopes. Priming with the peptide epitopes before infection with F. tularensis LVS increased the frequency of antigen-specific CD4 T cells as assessed by intracellular interferon-γ staining. These results illustrate the feasibility of screening an arrayed protein library that should be applicable to a variety of pathogens.

Interferon gamma and interleukin 4 have contrasting effects on immunopathology and the development of protective adaptive immunity against mycoplasma respiratory disease.

For vaccine development, it is critical to understand the regulatory mechanisms determining resistance and immunopathology against mycoplasma respiratory diseases. The present study evaluated the contribution of the polarizing cytokines interferon gamma (IFN-gamma) and interleukin 4 (IL-4) in the regulation of mycoplasma-specific immunity. The absence of a single cytokine (either IFN-gamma or IL-4) uniquely altered the expression of multiple chemokines and cytokines in the lungs of uninfected mice and influenced responses to mycoplasma infection. Most importantly, prior nasal-pulmonary immunization of IFN-gamma(-/-) mice led to exacerbated mycoplasma disease, whereas immunized IL-4(-/-) mice were dramatically more resistant than wild-type mice. Helper T cell type 2 responses in IFN-gamma(-/-) mice corresponded to immunopathologic reactions that developed after mycoplasma infection or immunization. Thus, adaptive immunity clearly can independently promote either protection or immunopathology against mycoplasma infection, and optimal vaccination appears to be dependent on promoting protective IFN-gamma-dependent networks (perhaps helper T cell type 1 responses) while minimizing the effect of IL-4-mediated responses, which dampen the generation of protective immunity.

Myeloid-associated lipin-1 transcriptional co-regulatory activity is atheroprotective.

BACKGROUND AND AIMS: Atherosclerosis is the most prominent underlying cause of cardiovascular disease (CVD). It is initiated by cholesterol deposition in the arterial intima, which causes macrophage recruitment and proinflammatory responses that promote plaque growth, necrotic core formation, and plaque rupture. Lipin-1 is a phosphatidic acid phosphohydrolase for glycerolipid synthesis. We have shown that lipin-1 phosphatase activity promotes macrophage pro-inflammatory responses when stimulated with modified low-density lipoprotein (modLDL) and accelerates atherosclerosis. Lipin-1 also independently acts as a transcriptional co-regulator where it enhances the expression of genes involved in ß-oxidation. In hepatocytes and adipocytes, lipin-1 augments the activity of transcription factors such as peroxisome proliferator-activated receptor (PPARs). PPARs control the expression of anti-inflammatory genes in macrophages and slow or reduce atherosclerotic progression. Therefore, we hypothesize myeloid-derived lipin-1 transcriptional co-regulatory activity reduces atherosclerosis. METHODS: We used myeloid-derived lipin-1 knockout (lipin-1mKO) and littermate control mice and AAV8-PCSK9 along with high-fat diet to elicit atherosclerosis. RESULTS: Lipin-1mKO mice had larger aortic root plaques than littermate control mice after 8 and 12 weeks of a high-fat diet. Lipin-1mKO mice also had increased serum proinflammatory cytokine concentrations, reduced apoptosis in plaques, and larger necrotic cores in the plaques compared to control mice. CONCLUSIONS: Combined, the data suggest lipin-1 transcriptional co-regulatory activity in myeloid cells is atheroprotective.

Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera.

The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week.IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.

Interface of Phospholipase Activity, Immune Cell Function, and Atherosclerosis.

Phospholipases are a family of lipid-altering enzymes that can either reduce or increase bioactive lipid levels. Bioactive lipids elicit signaling responses, activate transcription factors, promote G-coupled-protein activity, and modulate membrane fluidity, which mediates cellular function. Phospholipases and the bioactive lipids they produce are important regulators of immune cell activity, dictating both pro-inflammatory and pro-resolving activity. During atherosclerosis, pro-inflammatory and pro-resolving activities govern atherosclerosis progression and regression, respectively. This review will look at the interface of phospholipase activity, immune cell function, and atherosclerosis.

Macrophage-Associated Lipin-1 Promotes ß-Oxidation in Response to Proresolving Stimuli.

Macrophages reprogram their metabolism to promote appropriate responses. Proresolving macrophages primarily use fatty acid oxidation as an energy source. Metabolites generated during the catabolism of fatty acids aid in the resolution of inflammation and tissue repair, but the regulatory mechanisms that control lipid metabolism in macrophages are not fully elucidated. Lipin-1, a phosphatidic acid phosphatase that has transcriptional coregulator activity, regulates lipid metabolism in a variety of cells. In this current study, we show that lipin-1 is required for increased oxidative phosphorylation in IL-4 stimulated mouse (Mus musculus) macrophages. We also show that the transcriptional coregulatory function of lipin-1 is required for ß-oxidation in response to palmitate (free fatty acid) and apoptotic cell (human) stimulation. Mouse bone marrow-derived macrophages lacking lipin-1 have a reduction in critical TCA cycle metabolites following IL-4 stimulation, suggesting a break in the TCA cycle that is supportive of lipid synthesis rather than lipid catabolism. Together, our data demonstrate that lipin-1 regulates cellular metabolism in macrophages in response to proresolving stimuli and highlights the importance of aligning macrophage metabolism with macrophage phenotype.

Lipin-1 Contributes to IL-4 Mediated Macrophage Polarization.

Macrophage responses contribute to a diverse array of pathologies ranging from infectious disease to sterile inflammation. Polarization of macrophages determines their cellular function within biological processes. Lipin-1 is a phosphatidic acid phosphatase in which its enzymatic activity contributes to macrophage pro-inflammatory responses. Lipin-1 also possesses transcriptional co-regulator activity and whether this activity is required for macrophage polarization is unknown. Using mice that lack only lipin-1 enzymatic activity or both enzymatic and transcriptional coregulator activities from myeloid cells, we investigated the contribution of lipin-1 transcriptional co-regulator function toward macrophage wound healing polarization. Macrophages lacking both lipin-1 activities did not elicit IL-4 mediated gene expression to levels seen in either wild-type or lipin-1 enzymatically deficient macrophages. Furthermore, mice lacking myeloid-associated lipin-1 have impaired full thickness excisional wound healing compared to wild-type mice or mice only lacking lipin-1 enzymatic activity from myeloid cell. Our study provides evidence that lipin-1 transcriptional co-regulatory activity contributes to macrophage polarization and influences wound healing in vivo.