Multi-tissue profiling of oxylipins reveal a conserved up-regulation of epoxide:diol ratio that associates with white adipose tissue inflammation and liver steatosis in obesity.

BACKGROUND: Obesity drives maladaptive changes in the white adipose tissue (WAT) which can progressively cause insulin resistance, type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated liver disease (MASLD). Obesity-mediated loss of WAT homeostasis can trigger liver steatosis through dysregulated lipid pathways such as those related to polyunsaturated fatty acid (PUFA)-derived oxylipins. However, the exact relationship between oxylipins and metabolic syndrome remains elusive and cross-tissue dynamics of oxylipins are ill-defined. METHODS: We quantified PUFA-related oxylipin species in the omental WAT, liver biopsies and plasma of 88 patients undergoing bariatric surgery (female N = 79) and 9 patients (female N = 4) undergoing upper gastrointestinal surgery, using UPLC-MS/MS. We integrated oxylipin abundance with WAT phenotypes (adipogenesis, adipocyte hypertrophy, macrophage infiltration, type I and VI collagen remodelling) and the severity of MASLD (steatosis, inflammation, fibrosis) quantified in each biopsy. The integrative analysis was subjected to (i) adjustment for known risk factors and, (ii) control for potential drug-effects through UPLC-MS/MS analysis of metformin-treated fat explants ex vivo. FINDINGS: We reveal a generalized down-regulation of cytochrome P450 (CYP)-derived diols during obesity conserved between the WAT and plasma. Notably, epoxide:diol ratio, indicative of soluble epoxide hydrolyse (sEH) activity, increases with WAT inflammation/fibrosis, hepatic steatosis and T2DM. Increased 12,13-EpOME:DiHOME in WAT and liver is a marker of worsening metabolic syndrome in patients with obesity. INTERPRETATION: These findings suggest a dampened sEH activity and a possible role of fatty acid diols during metabolic syndrome in major metabolic organs such as WAT and liver. They also have implications in view of the clinical trials based on sEH inhibition for metabolic syndrome. FUNDING: Wellcome Trust (PS3431_WMIH); Duke-NUS (Intramural Goh Cardiovascular Research Award (Duke-NUS-GCR/2022/0020); National Medical Research Council (OFLCG22may-0011); National Institute of Environmental Health Sciences (Z01 ES025034); NIHR Imperial Biomedical Research Centre.

Systems level identification of a matrisome-associated macrophage polarisation state in multi-organ fibrosis.

Tissue fibrosis affects multiple organs and involves a master-regulatory role of macrophages which respond to an initial inflammatory insult common in all forms of fibrosis. The recently unravelled multi-organ heterogeneity of macrophages in healthy and fibrotic human disease suggests that macrophages expressing osteopontin (SPP1) associate with lung and liver fibrosis. However, the conservation of this SPP1+ macrophage population across different tissues and its specificity to fibrotic diseases with different etiologies remain unclear. Integrating 15 single-cell RNA-sequencing datasets to profile 235,930 tissue macrophages from healthy and fibrotic heart, lung, liver, kidney, skin, and endometrium, we extended the association of SPP1+ macrophages with fibrosis to all these tissues. We also identified a subpopulation expressing matrisome-associated genes (e.g., matrix metalloproteinases and their tissue inhibitors), functionally enriched for ECM remodelling and cell metabolism, representative of a matrisome-associated macrophage (MAM) polarisation state within SPP1+ macrophages. Importantly, the MAM polarisation state follows a differentiation trajectory from SPP1+ macrophages and is associated with a core set of regulon activity. SPP1+ macrophages without the MAM polarisation state (SPP1+MAM-) show a positive association with ageing lung in mice and humans. These results suggest an advanced and conserved polarisation state of SPP1+ macrophages in fibrotic tissues resulting from prolonged inflammatory cues within each tissue microenvironment.

Multinucleation resets human macrophages for specialized functions at the expense of their identity.

Macrophages undergo plasma membrane fusion and cell multinucleation to form multinucleated giant cells (MGCs) such as osteoclasts in bone, Langhans giant cells (LGCs) as part of granulomas or foreign-body giant cells (FBGCs) in reaction to exogenous material. How multinucleation per se contributes to functional specialization of mature mononuclear macrophages remains poorly understood in humans. Here, we integrate comparative transcriptomics with functional assays in purified mature mononuclear and multinucleated human osteoclasts, LGCs and FBGCs. Strikingly, in all three types of MGCs, multinucleation causes a pronounced downregulation of macrophage identity. We show enhanced lysosome-mediated intracellular iron homeostasis promoting MGC formation. The transition from mononuclear to multinuclear state is accompanied by cell specialization specific to each polykaryon. Enhanced phagocytic and mitochondrial function associate with FBGCs and osteoclasts, respectively. Moreover, human LGCs preferentially express B7-H3 (CD276) and can form granuloma-like clusters in vitro, suggesting that their multinucleation potentiates T cell activation. These findings demonstrate how cell-cell fusion and multinucleation reset human macrophage identity as part of an advanced maturation step that confers MGC-specific functionality.

The E3 ubiquitin ligase WWP2 regulates pro-fibrogenic monocyte infiltration and activity in heart fibrosis.

Non-ischemic cardiomyopathy (NICM) can cause left ventricular dysfunction through interstitial fibrosis, which corresponds to the failure of cardiac tissue remodeling. Recent evidence implicates monocytes/macrophages in the etiopathology of cardiac fibrosis, but giving their heterogeneity and the antagonizing roles of macrophage subtypes in fibrosis, targeting these cells has been challenging. Here we focus on WWP2, an E3 ubiquitin ligase that acts as a positive genetic regulator of human and murine cardiac fibrosis, and show that myeloid specific deletion of WWP2 reduces cardiac fibrosis in hypertension-induced NICM. By using single cell RNA sequencing analysis of immune cells in the same model, we establish the functional heterogeneity of macrophages and define an early pro-fibrogenic phase of NICM that is driven by Ccl5-expressing Ly6chigh monocytes. Among cardiac macrophage subtypes, WWP2 dysfunction primarily affects Ly6chigh monocytes via modulating Ccl5, and consequentially macrophage infiltration and activation, which contributes to reduced myofibroblast trans-differentiation. WWP2 interacts with transcription factor IRF7, promoting its non-degradative mono-ubiquitination, nuclear translocation and transcriptional activity, leading to upregulation of Ccl5 at transcriptional level. We identify a pro-fibrogenic macrophage subtype in non-ischemic cardiomyopathy, and demonstrate that WWP2 is a key regulator of IRF7-mediated Ccl5/Ly6chigh monocyte axis in heart fibrosis.

Immunolipidomics Reveals a Globoside Network During the Resolution of Pro-Inflammatory Response in Human Macrophages.

Toll-like receptor 4 (TLR4)-mediated changes in macrophages reshape intracellular lipid pools to coordinate an effective innate immune response. Although this has been previously well-studied in different model systems, it remains incompletely understood in primary human macrophages. Here we report time-dependent lipidomic and transcriptomic responses to lipopolysaccharide (LPS) in primary human macrophages from healthy donors. We grouped the variation of ~200 individual lipid species measured by LC-MS/MS into eight temporal clusters. Among all other lipids, glycosphingolipids (glycoSP) and cholesteryl esters (CE) showed a sharp increase during the resolution phase (between 8h or 16h post LPS). GlycoSP, belonging to the globoside family (Gb3 and Gb4), showed the greatest inter-individual variability among all lipids quantified. Integrative network analysis between GlycoSP/CE levels and genome-wide transcripts, identified Gb4 d18:1/16:0 and CE 20:4 association with subnetworks enriched for T cell receptor signaling (PDCD1, CD86, PTPRC, CD247, IFNG) and DC-SIGN signaling (RAF1, CD209), respectively. Our findings reveal Gb3 and Gb4 globosides as sphingolipids associated with the resolution phase of inflammatory response in human macrophages.

Glomerulonephritis and autoimmune vasculitis are independent of P2RX7 but may depend on alternative inflammasome pathways.

P2RX7, an ionotropic receptor for extracellular adenosine triphosphate (ATP), is expressed on immune cells, including macrophages, monocytes, and dendritic cells and is upregulated on nonimmune cells following injury. P2RX7 plays a role in many biological processes, including production of proinflammatory cytokines such as interleukin (IL)-1ß via the canonical inflammasome pathway. P2RX7 has been shown to be important in inflammation and fibrosis and may also play a role in autoimmunity. We have developed and phenotyped a novel P2RX7 knockout (KO) inbred rat strain and, taking advantage of the human-resembling unique histopathological features of rat models of glomerulonephritis, we induced three models of disease: nephrotoxic nephritis, experimental autoimmune glomerulonephritis, and experimental autoimmune vasculitis. We found that deletion of P2RX7 does not protect rats from models of experimental glomerulonephritis or the development of autoimmunity. Notably, treatment with A-438079, a P2RX7 antagonist, was equally protective in WKY WT and P2RX7 KO rats, revealing its 'off-target' properties. We identified a novel ATP/P2RX7/K+ efflux-independent and caspase-1/8-dependent pathway for the production of IL-1ß in rat dendritic cells, which was absent in macrophages. Taken together, these results comprehensively establish that inflammation and autoimmunity in glomerulonephritis is independent of P2RX7 and reveals the off-target properties of drugs previously known as selective P2RX7 antagonists. Rat mononuclear phagocytes may be able to utilise an 'alternative inflammasome' pathway to produce IL-1ß independently of P2RX7, which may account for the susceptibility of P2RX7 KO rats to inflammation and autoimmunity in glomerulonephritis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cardiac glycosides cause cytotoxicity in human macrophages and ameliorate white adipose tissue homeostasis.

BACKGROUND AND PURPOSE: Cardiac glycosides inhibit Na+ /K+ -ATPase and are used to treat heart failure and arrhythmias. They can induce inflammasome activation and pyroptosis in macrophages, suggesting cytotoxicity, which remains to be elucidated in human tissues. EXPERIMENTAL APPROACH: To determine the cell-type specificity of this cytotoxicity, we used human monocyte-derived macrophages and non-adherent peripheral blood cells from healthy donors, plus omental white adipose tissue, stromal vascular fraction-derived pre-adipocytes and adipocytes from obese patients undergoing bariatric surgery. All these cells/tissues were treated with nanomolar concentrations of ouabain (50, 100, 500 nM) to investigate the level of cytotoxicity and the mechanisms leading to cell death. In white adipose tissue, we investigated ouabain-mediated cytotoxicity by measuring insulin sensitivity, adipose tissue function and extracellular matrix deposition ex vivo. KEY RESULTS: Ouabain induced cell death through pyroptosis and apoptosis, and was more effective in monocyte-derived macrophages compared to non-adherent peripheral blood mononuclear cell populations. This cytotoxicity is dependent on K+ flux, as ouabain causes intracellular depletion of K+ and accumulation of Na+ and Ca2+ . Consistently, the cell death caused by these ion imbalances can be rescued by addition of potassium chloride to human monocyte-derived macrophages. Remarkably, when white adipose tissue explants from obese patients are cultured with nanomolar concentrations of ouabain, this causes depletion of macrophages, down-regulation of type VI collagen levels and amelioration of insulin sensitivity ex vivo. CONCLUSION AND IMPLICATIONS: The use of nanomolar concentration of cardiac glycosides could be an attractive therapeutic treatment for metabolic syndrome, characterized by pathogenic infiltration and activation of macrophages. LINKED ARTICLES: This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Adipoclast: a multinucleated fat-eating macrophage.

Cell membrane fusion and multinucleation in macrophages are associated with physiologic homeostasis as well as disease. Osteoclasts are multinucleated macrophages that resorb bone through increased metabolic activity resulting from cell fusion. Fusion of macrophages also generates multinucleated giant cells (MGCs) in white adipose tissue (WAT) of obese individuals. For years, our knowledge of MGCs in WAT has been limited to their description as part of crown-like structures (CLS) surrounding damaged adipocytes. However, recent evidence indicates that these cells can phagocytose oversized lipid remnants, suggesting that, as in osteoclasts, cell fusion and multinucleation are required for specialized catabolic functions. We thus reason that WAT MGCs can be viewed as functionally analogous to osteoclasts and refer to them in this article as adipoclasts. We first review current knowledge on adipoclasts and their described functions. In view of recent advances in single cell genomics, we describe WAT macrophages from a 'fusion perspective' and speculate on the ontogeny of adipoclasts. Specifically, we highlight the role of CD9 and TREM2, two plasma membrane markers of lipid-associated macrophages in WAT, which have been previously described as regulators of fusion and multinucleation in osteoclasts and MGCs. Finally, we consider whether strategies aiming to target WAT macrophages can be more selectively directed against adipoclasts.

Sphingolipid metabolism during Toll-like receptor 4 (TLR4)-mediated macrophage activation.

Macrophage activation in response to stimulation of Toll-like receptor 4 (TLR4) provides a paradigm for investigating energy metabolism that regulates the inflammatory response. TLR4-mediated pro-inflammatory macrophage activation is characterized by increased glycolysis and altered mitochondrial metabolism, supported by selective amino acid uptake and/or usage. Fatty acid metabolism remains as a highly complex rewiring that accompanies classical macrophage activation. TLR4 activation leads to de novo synthesis of fatty acids, which flux into sphingolipids, complex lipids that form the building blocks of eukaryotic cell membranes and regulate cell function. Here, we review the importance of TLR4-mediated de novo synthesis of membrane sphingolipids in macrophages. We first highlight fatty acid metabolism during TLR4-driven macrophage immunometabolism. We then focus on the temporal dynamics of sphingolipid biosynthesis and emphasize the modulatory role of some sphingolipid species (i.e. sphingomyelins, ceramides and glycosphingolipids) on the pro-inflammatory and pro-resolution phases of LPS/TLR4 activation in macrophages.

The versatile biochemistry of iron in macrophage effector functions.

Macrophages are mononuclear phagocytes with remarkable polarization ability that allow them to have tissue-specific functions during development, homeostasis, inflammatory and infectious disease. One particular trophic factor in the tissue environment is iron, which is intimately linked to macrophage effector functions. Macrophages have a well-described role in the control of systemic iron levels, but their activation state is also depending on iron-containing proteins/enzymes. Haemoproteins, dioxygenases and iron-sulphur (Fe-S) enzymes are iron-binding proteins that have bactericidal, metabolic and epigenetic-related functions, essential to shape the context-dependent macrophage polarization. In this review, I describe mainly pro-inflammatory macrophage polarization focussing on the role of iron biochemistry in selected haemoproteins and Fe-S enzymes. I show how iron, as part of haem or Fe-S clusters, participates in the cellular control of pro-inflammatory redox reactions in parallel with its role as enzymatic cofactor. I highlight a possible coordinated regulation of haemoproteins and Fe-S enzymes during classical macrophage activation. Finally, I describe tryptophan and α-ketoglutarate metabolism as two essential effector pathways in macrophages that use diverse iron biochemistry at different enzymatic steps. Through these pathways, I show how iron participates in the regulation of essential metabolites that shape macrophage function.

Similarities and interplay between senescent cells and macrophages.

Senescence is a cellular program that prevents the replication of old, damaged, or cancerous cells. Senescent cells become growth arrested and undergo changes in their morphology, chromatin organization, and metabolism, and produce a bioactive secretome. This secretome, the senescence-associated secretory phenotype (SASP), mediates many of the pathophysiological effects associated with senescent cells, for example, recruiting and activating immune cells such as macrophages. The relation between senescent cells and macrophages is intriguing: senescent cells recruit macrophages, can induce them to undergo senescence, or can influence their polarization. Senescent cells and macrophages share multiple phenotypic characteristics; both have a high secretory status, increased lysosome numbers, or the ability to activate the inflammasome. Senescent cells accumulate during aging and disease, and killing them results in widespread benefits. Here we discuss similarities between senescent cells and macrophages and interpret the latest developments in macrophage biology to understand the molecular mechanisms of cellular senescence. We describe evidence and effects of senescence in macrophages and speculate on the ontogeny of the senescent-like state in macrophages. Finally, we examine the macrophage-senescent cell interplay and its impact on macrophage effector functions during inflammatory conditions and in the tumor microenvironment.

BCAT1 affects mitochondrial metabolism independently of leucine transamination in activated human macrophages.

In response to environmental stimuli, macrophages change their nutrient consumption and undergo an early metabolic adaptation that progressively shapes their polarization state. During the transient, early phase of pro-inflammatory macrophage activation, an increase in tricarboxylic acid (TCA) cycle activity has been reported, but the relative contribution of branched-chain amino acid (BCAA) leucine remains to be determined. Here, we show that glucose but not glutamine is a major contributor of the increase in TCA cycle metabolites during early macrophage activation in humans. We then show that, although uptake of BCAAs is not altered, their transamination by BCAT1 is increased following 8 h lipopolysaccharide (LPS) stimulation. Of note, leucine is not metabolized to integrate into the TCA cycle in basal or stimulated human macrophages. Surprisingly, the pharmacological inhibition of BCAT1 reduced glucose-derived itaconate, α-ketoglutarate and 2-hydroxyglutarate levels without affecting succinate and citrate levels, indicating a partial inhibition of the TCA cycle. This indirect effect is associated with NRF2 (also known as NFE2L2) activation and anti-oxidant responses. These results suggest a moonlighting role of BCAT1 through redox-mediated control of mitochondrial function during early macrophage activation.

Cardiac glycosides are broad-spectrum senolytics.

Senescence is a cellular stress response that results in the stable arrest of old, damaged or preneoplastic cells. Oncogene-induced senescence is tumor suppressive but can also exacerbate tumorigenesis through the secretion of pro-inflammatory factors from senescent cells. Drugs that selectively kill senescent cells, termed senolytics, have proved beneficial in animal models of many age-associated diseases. Here, we show that the cardiac glycoside, ouabain, is a senolytic agent with broad activity. Senescent cells are sensitized to ouabain-induced apoptosis, a process mediated in part by induction of the pro-apoptotic Bcl2-family protein NOXA. We show that cardiac glycosides synergize with anti-cancer drugs to kill tumor cells and eliminate senescent cells that accumulate after irradiation or in old mice. Ouabain also eliminates senescent preneoplastic cells. Our findings suggest that cardiac glycosides may be effective anti-cancer drugs by acting through multiple mechanism. Given the broad range of senescent cells targeted by cardiac glycosides their use against age-related diseases warrants further exploration.

Acute Iron Deprivation Reprograms Human Macrophage Metabolism and Reduces Inflammation In Vivo.

Iron is an essential metal that fine-tunes the innate immune response by regulating macrophage function, but an integrative view of transcriptional and metabolic responses to iron perturbation in macrophages is lacking. Here, we induced acute iron chelation in primary human macrophages and measured their transcriptional and metabolic responses. Acute iron deprivation causes an anti-proliferative Warburg transcriptome, characterized by an ATF4-dependent signature. Iron-deprived human macrophages show an inhibition of oxidative phosphorylation and a concomitant increase in glycolysis, a large increase in glucose-derived citrate pools associated with lipid droplet accumulation, and modest levels of itaconate production. LPS polarization increases the itaconate:succinate ratio and decreases pro-inflammatory cytokine production. In rats, acute iron deprivation reduces the severity of macrophage-dependent crescentic glomerulonephritis by limiting glomerular cell proliferation and inducing lipid accumulation in the renal cortex. These results suggest that acute iron deprivation has in vivo protective effects mediated by an anti-inflammatory immunometabolic switch in macrophages.

Common signalling pathways in macrophage and osteoclast multinucleation.

Macrophage cell fusion and multinucleation are fundamental processes in the formation of multinucleated giant cells (MGCs) in chronic inflammatory disease and osteoclasts in the regulation of bone mass. However, this basic cell phenomenon is poorly understood despite its pathophysiological relevance. Granulomas containing multinucleated giant cells are seen in a wide variety of complex inflammatory disorders, as well as in infectious diseases. Dysregulation of osteoclastic bone resorption underlies the pathogenesis of osteoporosis and malignant osteolytic bone disease. Recent reports have shown that the formation of multinucleated giant cells and osteoclast fusion display a common molecular signature, suggesting shared genetic determinants. In this Review, we describe the background of cell-cell fusion and the similar origin of macrophages and osteoclasts. We specifically focus on the common pathways involved in osteoclast and MGC fusion. We also highlight potential approaches that could help to unravel the core mechanisms underlying bone and granulomatous disorders in humans.

Changes in macrophage transcriptome associate with systemic sclerosis and mediate GSDMA contribution to disease risk.

OBJECTIVES: Several common and rare risk variants have been reported for systemic sclerosis (SSc), but the effector cell(s) mediating the function of these genetic variants remains to be elucidated. While innate immune cells have been proposed as the critical targets to interfere with the disease process underlying SSc, no studies have comprehensively established their effector role. Here we investigated the contribution of monocyte-derived macrophages (MDMs) in mediating genetic susceptibility to SSc. METHODS: We carried out RNA sequencing and genome-wide genotyping in MDMs from 57 patients with SSc and 15 controls. Our differential expression and expression quantitative trait locus (eQTL) analysis in SSc was further integrated with epigenetic, expression and eQTL data from skin, monocytes, neutrophils and lymphocytes. RESULTS: We identified 602 genes upregulated and downregulated in SSc macrophages that were significantly enriched for genes previously implicated in SSc susceptibility (P=5×10-4), and 270 cis-regulated genes in MDMs. Among these, GSDMA was reported to carry an SSc risk variant (rs3894194) regulating expression of neighbouring genes in blood. We show that GSDMA is upregulated in SSc MDMs (P=8.4×10-4) but not in the skin, and is a significant eQTL in SSc macrophages and lipopolysaccharide/interferon gamma (IFNγ)-stimulated monocytes. Furthermore, we identify an SSc macrophage transcriptome signature characterised by upregulation of glycolysis, hypoxia and mTOR signalling and a downregulation of IFNγ response pathways. CONCLUSIONS: Our data further establish the link between macrophages and SSc, and suggest that the contribution of the rs3894194 risk variant to SSc susceptibility can be mediated by GSDMA expression in macrophages.

Identification of Ceruloplasmin as a Gene that Affects Susceptibility to Glomerulonephritis Through Macrophage Function.

Crescentic glomerulonephritis (Crgn) is a complex disorder where macrophage activity and infiltration are significant effector causes. In previous linkage studies using the uniquely susceptible Wistar Kyoto (WKY) rat strain, we have identified multiple crescentic glomerulonephritis QTL (Crgn) and positionally cloned genes underlying Crgn1 and Crgn2, which accounted for 40% of total variance in glomerular inflammation. Here, we have generated a backcross (BC) population (n = 166) where Crgn1 and Crgn2 were genetically fixed and found significant linkage to glomerular crescents on chromosome 2 (Crgn8, LOD = 3.8). Fine mapping analysis by integration with genome-wide expression QTLs (eQTLs) from the same BC population identified ceruloplasmin (Cp) as a positional eQTL in macrophages but not in serum. Liquid chromatography-tandem mass spectrometry confirmed Cp as a protein QTL in rat macrophages. WKY macrophages overexpress Cp and its downregulation by RNA interference decreases markers of glomerular proinflammatory macrophage activation. Similarly, short incubation with Cp results in a strain-dependent macrophage polarization in the rat. These results suggest that genetically determined Cp levels can alter susceptibility to Crgn through macrophage function and propose a new role for Cp in early macrophage activation.

Macrophage epoxygenase determines a profibrotic transcriptome signature.

Epoxygenases belong to the cytochrome P450 family. They generate epoxyeicosatrienoic acids, which are known to have anti-inflammatory effects, but little is known about their role in macrophage function. By high-throughput sequencing of RNA in primary macrophages derived from rodents and humans, we establish the relative expression of epoxygenases in these cells. Zinc-finger nuclease-mediated targeted gene deletion of the major rat macrophage epoxygenase Cyp2j4 (ortholog of human CYP2J2) resulted in reduced epoxyeicosatrienoic acid synthesis. Cyp2j4(-/-) macrophages have relatively increased peroxisome proliferator-activated receptor-γ levels and show a profibrotic transcriptome, displaying overexpression of a specific subset of genes (260 transcripts) primarily involved in extracellular matrix, with fibronectin being the most abundantly expressed transcript. Fibronectin expression is under the control of epoxygenase activity in human and rat primary macrophages. In keeping with the in vitro findings, Cyp2j4(-/-) rats show upregulation of type I collagen following unilateral ureter obstruction of the kidney, and quantitative proteomics analysis (liquid chromatography-tandem mass spectrometry) showed increased renal type I collagen and fibronectin protein abundance resulting from experimentally induced crescentic glomerulonephritis in these rats. Taken together, these results identify the rat epoxygenase Cyp2j4 as a determinant of a profibrotic macrophage transcriptome that could have implications in various inflammatory conditions, depending on macrophage function.

Use of Quantitative Real Time Polymerase Chain Reaction to Assess Gene Transcripts Associated With Antibody-Mediated Rejection of Kidney Transplants.

INTRODUCTION: Microarray studies have shown elevated transcript levels of endothelial and natural killer (NK) cell-associated genes during antibody-mediated rejection (AMR) of the renal allograft. This study aimed to assess the use of quantitative real-time polymerase chain reaction as an alternative to microarray analysis on a subset of these elevated genes. METHODS: Thirty-nine renal transplant biopsies from patients with de novo donor-specific antibodies and eighteen 1-year surveillance biopsies with no histological evidence of rejection were analyzed for expression of 11 genes previously identified as elevated in AMR. RESULTS: Expression levels of natural killer markers were correlated to microcirculation inflammation and graft outcomes to a greater extent than endothelial markers. Creating a predictive model reduced the number of gene transcripts to be assessed to 2, SH2D1b and MYBL1, resulting in 66.7% sensitivity and 89.7% specificity for graft loss. DISCUSSION: This work demonstrates that elevated gene expression levels, proposed to be associated with AMR, can be detected by established quantitative real-time polymerase chain reaction technology, making transition to the clinical setting feasible. Transcript analysis provides additional diagnostic information to the classification schema for AMR diagnosis but it remains to be determined whether significant numbers of centres will validate transcript analysis in their laboratories and put such analysis into clinical use.