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.

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.

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.

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 COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 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 inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVΔG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

AAV8-mediated overexpression of mPCSK9 in liver differs between male and female mice.

BACKGROUND AND AIMS: The recombinant adeno-associated viral vector serotype 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (AAV8- PCSK9) is a new model for the induction of hypercholesterolemia. AAV8 preferentially infects hepatocytes and the incorporated liver-specific promoter should ensure expression of PCSK9 in the liver. Since tissue distribution of AAVs can differ between male and female mice, we investigated the differences in PCSK9 expression and hypercholesterolemia development between male and female mice using the AAV8-PCSK9 model. METHODS: Male and female C57BL/6 mice were injected with either a low-dose or high-dose of AAV8-PCSK9 and fed a high-fat diet. Plasma lipid levels were evaluated as a measure of the induction of hypercholesterolemia. RESULTS: Injection of mice with low dose AAV8-PCSK9 dramatically elevated both serum PCSK9 and cholesterol levels in male but not female mice. Increasing the dose of AAV8-PCSK9 threefold in female mice rescued the hypercholesterolemia phenotype but did not result in full restoration of AAV8-PCSK9 transduction of livers in female mice compared to the low-dose male mice. Our data demonstrate female mice respond differently to AAV8-PCSK9 injection compared to male mice. CONCLUSIONS: These differences do not hinder the use of female mice when AAV8-PCSK9 doses are taken into consideration. However, localization to and production of AAV8-PCSK9 in organs besides the liver in mice may introduce confounding factors into studies and should be considered during experimental design.

Gene Transfer Induced Hypercholesterolemia in Amyloid Mice.

A risk factor for cardiovascular disease (CVD), mutant PCSK9, was expressed in APP/PS1 mice to study the CVD-Alzheimer's disease inter-relationship. Cholesterol levels were elevated by 5-6-fold from 3 to 13 weeks after PCSK9 gene transfer. We tested whether hypercholesterolemia would increase amyloid-ß plaques at a relatively early stage of plaque deposition. Plaque burden was increased in the hippocampus of PCSK9 treated mice though the increase was modest compared to the large elevation in cholesterol. Elevating cholesterol via gene transfer could be valuable in a variety of disease models compared to making crosses with germ-line transgenic mouse models of CVD.