key: cord-0285451-cvpm89o1 authors: Ryan, Dylan G.; Knatko, Elena V.; Casey, Alva; Hukelmann, Jens L.; Brenes, Alejandro J.; Naidu, Sharadha Dayalan; Higgins, Maureen; Tronci, Laura; Nikitopoulou, Efterpi; O’Neill, Luke A.J.; Frezza, Christian; Lamond, Angus I.; Abramov, Andrey Y.; Cantrell, Doreen A.; Murphy, Michael P.; Dinkova-Kostova, Albena T. title: Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response date: 2021-08-13 journal: bioRxiv DOI: 10.1101/2021.08.13.456204 sha: b71ca763dba753455f47bcb524a9ff9bd1d0de80 doc_id: 285451 cord_uid: cvpm89o1 To overcome oxidative, inflammatory, and metabolic stress, cells have evolved networks of cytoprotective proteins controlled by nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its main negative regulator the Kelch-like ECH associated protein 1 (Keap1). Here, we used high-resolution mass-spectrometry to characterize the proteomes of macrophages with genetically altered Nrf2 status. Our analysis revealed significant differences among the genotypes in cellular metabolism and redox homeostasis, which we validated with respirometry and metabolomics, as well as in anti-viral immune pathways and the cell cycle. Nrf2 status significantly affected the proteome following lipopolysaccharide (LPS) stimulation, with alterations in redox, carbohydrate and lipid metabolism, and innate immunity observed. Of note, Nrf2 activation was found to promote mitochondrial fusion in inflammatory macrophages. The Keap1 inhibitor, 4-octyl itaconate (4-OI), a derivative of the mitochondrial immunometabolite itaconate, remodeled the inflammatory macrophage proteome, increasing redox and suppressing anti-viral immune effectors in a Nrf2-dependent manner. These data suggest that Nrf2 activation facilitates metabolic reprogramming and mitochondrial adaptation, and finetunes the innate immune response in macrophages. Graphical abstract Highlights First high-resolution proteome of macrophages with genetically altered Nrf2 status Nrf2 is key regulator of macrophage redox and intermediary metabolism Nrf2 finetunes the inflammatory response suppressing anti-viral immune and cytokine effectors, whilst promoting T cell activation factors Nrf2 regulates mitochondrial adaptation in inflammatory macrophages promoting the formation of a fused network 4-octyl itaconate (4-OI) suppresses anti-viral immune effectors in inflammatory macrophages in a Nrf2-dependent manner Nrf2, Nfe2l2, Keap1, LPS, 4-octyl itaconate, macrophage, immunometabolism, 49 mitochondria, mitochondrial dynamics, innate immunity, cytokine, anti-viral immune 50 response, mass spectrometry, proteomics 51 52 Introduction 66 67 The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2, gene name 68 6 decreased intracellular GSH, taurine, hypotaurine and -alanine ( Figure 2C) . Indeed, 176 the rate-limiting GSH biosynthetic enzymes, Gclc and Gclm, as well as Nqo1 and Gsr, 177 were decreased with Nrf2-KO and increased with Keap1-KD ( Figure S2F) . 178 Furthermore, both Nrf2 activation and disruption led to significant alterations in 179 mitochondrial metabolites, such as those involved in fatty acid oxidation (FAO) 180 (carnitine, palmitoylcarnitine, hexanoylcarnitine and tetradecanoylcarnitine), the TCA 181 cycle (fumarate, malate and 2-ketoglutarate) and bioenergetics (NAD, creatine, 182 phosphocreatine). These findings suggest an involvement of Nrf2 in regulating 183 mitochondrial metabolism in macrophages. 184 To confirm these observations using MS-independent methods, we performed 185 respirometry analysis of oxygen consumption rates (OCR) in all three genotypes. This 186 identified a role for Nrf2 in regulating mitochondrial respiration ( Figure 2D cluster that includes a plethora of intermediary metabolic pathways associated with 205 carbohydrate, cofactor and energy metabolism, and a smaller cluster for the cellular 206 response to oxidative stress ( Figure 3A) . The most significantly enriched pathways 207 included those involved in glycolysis and GSH metabolism ( Figure S3C ). In Keap1-208 KD positively regulated processes, a loosely interconnected functional cluster was 209 observed with an enrichment in processes associated with lipid metabolism, amino 210 acid metabolism and cofactor metabolism, while enrichment in regulators of reactive 211 oxygen species (ROS), cell adhesion and organic ion transport were also observed 212 ( Figure 3B) . The most significantly enriched pathways included those related to lipid 213 metabolism, such as FAO, and ROS detoxification ( Figure S3D ). In contrast, we did 214 not observe many significant increases in the proteome of LPS-treated Nrf2-KO 215 macrophages with no clearly regulated pathways (Figure S3A) , whilst a large 216 functional cluster was associated with the regulation of T cell activation in the 217 decreased targets of Keap1-KD macrophages ( Figure S3B) . as the cut-off. For proteomics data, protein copy number was converted to a log2 scale 516 and biological replicates were grouped by experimental condition. Protein-wise linear 517 models combined with empirical Bayes statistics were used for the differential 518 expression analyses. The Bioconductor package limma was used to carry out the 519 analysis using an R based online tool (Shah et al., 2020) . Data were visualized using 520 a volcano plot, which shows the log2 fold change on the x axis and the adjusted p value 521 on the y axis. Overrepresentation analysis (ORA) of significant changes were 522 assessed using Enrichr (Kuleshov et al., 2016) MaxQuant enables high peptide identification rates, 661 individualized p.p.b.-range mass accuracies and proteome-wide protein 662 quantification Can Activation of NRF2 Be a Strategy COVID-19? Nrf2 is activated 669 by disruption of mitochondrial thiol homeostasis but not by enhanced 670 mitochondrial superoxide production Activation of the Antiviral Kinase PKR and Viral 672 Countermeasures C151 in KEAP1 675 is the main cysteine sensor for the cyanoenone class of NRF2 activators, 676 irrespective of molecular size or shape Targeting Nrf2 signaling 679 improves bacterial clearance by alveolar macrophages in patients with COPD and 680 in a mouse model Nrf2 impacts cellular bioenergetics by controlling substrate availability for 687 mitochondrial respiration Quantitative analysis of T cell proteomes and 690 environmental sensors during T cell differentiation Role of the ISR-ATF4 pathway and its cross talk with Nrf2 in mitochondrial 693 quality control Nrf2 Activation Protects 696 against Solar-Simulated Ultraviolet Radiation in Mice and Humans Keap1 Confers Features of a Fasted Metabolic State Nrf2 suppresses macrophage inflammatory response by blocking 704 proinflammatory cytokine transcription Enrichr: a 707 comprehensive gene set enrichment analysis web server 2016 update 4-Octyl Itaconate Alleviates Lipopolysaccharide-Induced Acute Lung 711 Injury in Mice by Inhibiting Oxidative Stress and Inflammation. Drug design, 712 development Itaconate ameliorates methicillin-resistant Staphylococcus 715 aureus-induced acute lung injury through the Nrf2/ARE pathway Med 9 Biomarker Exploration in Human 719 Peripheral Blood Mononuclear Cells for Monitoring Sulforaphane Treatment 720 Responses in Nrf2 affects the efficiency of mitochondrial fatty acid oxidation Nrf2 regulates the 726 alternative first exons of CD36 in macrophages through specific antioxidant 727 response elements Itaconate is 734 an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. 735 Nrf2 negatively 738 regulates STING indicating a link between antiviral sensing and metabolic 739 reprogramming SARS-CoV2-mediated 742 suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory 743 activity of 4-octyl-itaconate and dimethyl fumarate Augments Vesicular Stomatitis Virus Oncolysis via Autophagy-Driven 748 Suppression of Antiviral Immunity Sulforaphane exhibits in vitro and in vivo antiviral activity against pandemic 752 SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses MetaboAnalyst 5.0: narrowing the gap 755 between raw spectra and functional insights The 758 PRIDE database and related tools and resources in 2019: improving support for 759 quantification data Coupling Krebs cycle metabolites to signalling in 762 immunity and cancer Krebs Cycle Reborn in Macrophage 764 Nrf2 Induces IL-17D to Mediate Tumor and Virus Surveillance LFQ-Analyst: An Easy-To-Use Interactive Web Platform To Analyze and Visualize 771 Proteomics Data Preprocessed with MaxQuant The intracellular 774 redox state is a core determinant of mitochondrial fusion The Perseus computational platform for comprehensive analysis 784 of (prote)omics data Metabolic 786 Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis A "proteomic ruler" for 789 protein copy number and concentration estimation without spike Single-cell 793 RNA-sequencing of herpes simplex virus 1-infected cells connects NRF2 794 activation to an antiviral program System: a Thiol-Based Sensor-Effector Apparatus for Maintaining Redox 797 Four-octyl itaconate activates Nrf2 cascade to protect osteoblasts from 800 hydrogen peroxide-induced oxidative injury