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The present study explores the role of the cytosolic branched chain amino acid aminotransferase (BCAT1) in CD8+T cell activation, in general, and tumor immunity, in particular, and identifies a non-canonical function of the protein in iron homeostasis. Pharmacologic inhibition of BCAT1 using the novel drug ERG245 abrogates the effector functions of CD8+T cells in vitro and metabolically reprograms the cells towards increased OXPHOS. In vivo, it suppresses activation of CD8+T cells in DSS colitis leading to improved disease outcomes. Remarkably, withdrawal of BCAT1 inhibition further amplifies OXPHOS and gives rise to CD8+T cells with increased cytotoxicity in vitro and in vivo. When combined with an anti-PD-1 treatment, temporal BCAT1 inhibition dramatically increases anti-PD-1 efficacy inducing complete and durable tumor regressions in the moderately immunogenic CT26 tumor model. Single cell RNA-seq data link expression of Bcat genes to exhausted T cells within the tumor microenvironment of human cancer patients, whereas in vitro assays indicate that BCAT1 inhibition partially prevents the adoption of a terminally exhausted phenotype by CD8+T cells. We propose BCAT1 as a target for cancer combinatory therapies.SIGNIFICANCEThe study explores for the first time the role of BCAT1 in CD8+T cell activation and proposes novel strategies for using BCAT1 inhibitors in cancer and beyond. It demonstrates that BCAT1 exerts its function without significantly altering branched chain amino acid (BCAA) levels through a mechanism that controls iron homeostasis, a novel non-canonical mechanism of action, and implicates BCAT1 in the adoption of an exhausted phenotype by T cells found in human cancers. While the majority of metabolic drugs temper OXPHOS, it demonstrates that an agent that increases OXPHOS in CD8+T cells can be used successfully as an immune-oncology drug.

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 unraveled multi-organ heterogeneity of macrophages in healthy and fibrotic human disease suggest that tissue resident 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 13 single cell RNA-sequencing datasets to profile 225,985 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 remodeling and cell metabolism, representative of a matrisome-associated macrophage (MAM) polarization state within SPP1+ macrophages. Importantly, the MAM polarization state follows a differentiation trajectory from SPP1+ macrophages, which was conserved across all fibrotic tissues and driven by NFATC1 and HIVEP3 regulons. Unlike SPP1+ macrophages, the MAM polarization state shows a positive association with ageing in mice and humans, and across multiple tissues during homeostasis. These results suggest an advanced, age-dependent polarization state of SPP1+ macrophages in fibrotic tissues as a result of prolonged inflammatory cues within each tissue microenvironment.

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.

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. While osteoclast multinucleation is a prerequisite for vertebrate bone homeostasis, the effector function resulting from LGC and FBGC multinucleation is less well-defined. More generally, how multinucleation per se contributes to functional specialization of mature mononuclear macrophages remains poorly understood in humans. Here, we integrated 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 down-regulation of mononuclear phagocyte 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, only B7-H3 (CD276)-expressing human LGCs can form granuloma-like clusters in vitro, suggesting that LGC 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.

Background and purpose Cardiac glycosides (CGs) inhibit the Na+ ,K+ -ATPase and are widely prescribed medicines for chronic heart failure and cardiac arrhythmias. Recently, CGs have been described to induce inflammasome activation and pyroptosis in human macrophages, suggesting a cytotoxicity that remains to be elucidated in tissues. Experimental approach To determine the cell type specificity of CG-mediated cytotoxicity, we used human primary monocyte-derived macrophages (hMDMs) and non-adherent peripheral blood cells isolated from healthy donors. Omental white adipose tissue (WAT) and stromal vascular fraction (SVF)-derived pre-adipocytes and adipocytes were isolated from obese patients undergoing bariatric surgery. All these primary cells/tissues were treated with nanomolar concentrations of ouabain (50nM, 100nM and 500nM) to investigate its degree of cytotoxicity and mechanisms leading to cell death. In WAT, we further explored the consequences of ouabain-mediated cytotoxicity by measuring insulin sensitivity, adipose tissue function and extracellular matrix (ECM) deposition ex vivo. Key results The ouabain-induced cell death is through pyroptosis and apoptosis, and more efficient in hMDMs compared to non-adherent PBMC populations. This selective cytotoxicity is dependent on K+ flux, as ouabain causes an intracellular depletion of K+ , while inducing accumulation of Na+ and Ca2+ levels. Consistently, the cell-death caused by these ion imbalances can be rescued by addition of potassium chloride in hMDMs. Remarkably, when WAT 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. Conclusions and implications These results suggest that the usage of nanomolar concentration of CGs can be an attractive therapeutic avenue in metabolic syndrome characterised by pathogenic infiltration and activation of 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.

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.

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.

Behmoaras and Gil describe shared cellular features between senescent cells and macrophages and outline the interaction between the two cell types during homeostasis and disease., 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.

End-stage kidney disease (ESKD) patients are at high risk of severe COVID-19. We performed dense serial blood sampling in hospitalised and non-hospitalised ESKD patients with COVID-19 (n=256 samples from 55 patients) and used Olink immunoassays to measure 436 circulating proteins. Comparison to 51 non-infected ESKD patients revealed 221 proteins differentially expressed in COVID-19, of which 69.7% replicated in an independent cohort of 46 COVID-19 patients. 203 proteins were associated with clinical severity scores, including IL6, markers of monocyte recruitment (e.g. CCL2, CCL7), neutrophil activation (e.g proteinase-3) and epithelial injury (e.g. KRT19). Random Forests machine learning identified predictors of current or future severity such as KRT19, PARP1, PADI2, CCL7, and IL1RL1 (ST2). Survival analysis with joint models revealed 69 predictors of death including IL22RA1, CCL28, and the neutrophil-derived chemotaxin AZU1 (Azurocidin). Finally, longitudinal modelling with linear mixed models uncovered 32 proteins that display different temporal profiles in severe versus non-severe disease, including integrins and adhesion molecules. Our findings point to aberrant innate immune activation and leucocyte-endothelial interactions as central to the pathology of severe COVID-19. The data from this unique cohort of high-risk individuals provide a valuable resource for identifying drug targets in COVID-19.

Cardiac glycosides (CGs) inhibit the Na+,K+-ATPase and are widely prescribed medicines for chronic heart failure and cardiac arrhythmias. Recently, CGs have been described to induce inflammasome activation in human macrophages, suggesting a cytotoxicity that remains to be elucidated in tissues. Here we show that human monocyte-derived macrophages (hMDMs) undergo cell death following incubation with nanomolar concentrations of CGs, and in particular with ouabain (IC50=50 nM). The ouabain-induced cell death is more efficient in hMDMs compared to non-adherent PBMC populations and is through on-target inhibition of Na,K-ATPAse activity, as it causes an intracellular depletion of K+, while inducing accumulation of Na+ and Ca2+ levels. Consistently, the cell-death caused by these ion imbalances can be rescued by addition of potassium chloride in hMDMs. Strikingly, white adipose tissue (WAT) explants from morbidly obese patients cultured with nanomolar concentrations of ouabain causes depletion of macrophages, decreases type VI collagen levels, and ameliorates insulin-sensitivity ex vivo. These results suggest that the usage of nanomolar concentration of CGs can be an attractive therapeutic avenue in metabolic syndrome characterised by pathogenic infiltration and activation of macrophages.

The majority of patients with systemic lupus erythematosus (SLE) have high expression of type I IFN-stimulated genes. Mitochondrial abnormalities have also been reported, but the contribution of type I IFN exposure to these changes is unknown. Here, we show downregulation of mitochondria-derived genes and mitochondria-associated metabolic pathways in IFN-High patients from transcriptomic analysis of CD4

Functional characterisation of cell-type specific regulatory networks is key to establish a causal link between genetic variation and phenotype. The osteoclast offers a unique model for interrogating the contribution of co-regulated genes to in vivo phenotype as its multinucleation and resorption activities determine quantifiable skeletal traits. Here we took advantage of a trans-regulated gene network (MMnet, macrophage multinucleation network) which we found to be significantly enriched for GWAS variants associated with bone-related phenotypes. We found that the network hub gene Bcat1 and seven other co-regulated MMnet genes out of 13, regulate bone function. Specifically, global (Pik3cb−/−, Atp8b2+/−, Igsf8−/−, Eml1−/−, Appl2−/−, Deptor−/−) and myeloid-specific Slc40a1ΔLysMCre knockout mice displayed abnormal bone phenotypes. We report antagonizing effects of MMnet genes on bone mass in mice and osteoclast multinucleation/resorption in humans with strong correlation between the two. These results identify MMnet as a functionally conserved network that regulates osteoclast fusion and bone mass.Impact statementWe took advantage of the osteoclast whose multinucleation properties correlate with bone mass. We show that a trans-regulated gene network (MMnet) controls skeletal homeostasis through osteoclast multinucleation and function.

International audience; Functional characterisation of cell-type-specific regulatory networks is key to establish a causal link between genetic variation and phenotype. The osteoclast offers a unique model for interrogating the contribution of co-regulated genes to in vivo phenotype as its multinucleation and resorption activities determine quantifiable skeletal traits. Here we took advantage of a trans-regulated gene network (MMnet, macrophage multinucleation network) which we found to be significantly enriched for GWAS variants associated with bone-related phenotypes. We found that the network hub gene Bcat1 and seven other co-regulated MMnet genes out of 13, regulate bone function. Specifically, global (Pik3cb-/-, Atp8b2+/-, Igsf8-/-, Eml1-/-, Appl2-/-, Deptor-/-) and myeloid-specific Slc40a1 knockout mice displayed abnormal bone phenotypes. We report opposing effects of MMnet genes on bone mass in mice and osteoclast multinucleation/resorption in humans with strong correlation between the two. These results identify MMnet as a functionally conserved network that regulates osteoclast multinucleation and bone mass.

Senescence is a cellular stress response that results in the stable arrest of old, damaged or pre-neoplastic cells. Oncogene-induced senescence is tumour suppressive but can also exacerbate tumorigenesis through the secretion of proinflammatory factors from senescent cells. Drugs that selectively kill senescent cells, termed ‘senolytics’, have proved beneficial in animal models of many age-associated diseases. In the present study, 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 proapoptotic Bcl-2 family protein NOXA. We demonstrate that cardiac glycosides synergize with anti-cancer drugs to kill tumour cells and eliminate senescent cells that accumulate after irradiation or in old mice. Ouabain also eliminates senescent pre-neoplastic cells. The findings of the present study suggest that cardiac glycosides may be effective anti-cancer drugs by acting through multiple mechanisms. Given the broad range of senescent cells targeted by cardiac glycosides, their use against age-related diseases warrants further exploration.

Among other cells, macrophages regulate the inflammatory and reparative phases during wound healing but genetic determinants and detailed molecular pathways that modulate these processes are not fully elucidated. Here, we took advantage of normal variation in wound healing in 1,378 genetically outbred mice, and carried out macrophage RNA-sequencing profiling of mice with extreme wound healing phenotypes (i.e., slow and fast healers, n = 146 in total). The resulting macrophage coexpression networks were genetically mapped and led to the identification of a unique module under strong trans-acting genetic control by the Runx2 locus. This macrophage-mediated healing network was specifically enriched for cholesterol and fatty acid biosynthetic processes. Pharmacological blockage of fatty acid synthesis with cerulenin resulted in delayed wound healing in vivo, and increased macrophage infiltration in the wounded skin, suggesting the persistence of an unresolved inflammation. We show how naturally occurring sequence variation controls transcriptional networks in macrophages, which in turn regulate specific metabolic pathways that could be targeted in wound healing.

BCAT1, the enzyme responsible for the reversible transamination of leucine in the cytosol, has recently been implicated in the development and growth of various types of cancer. The limited expression of BCAT1 in adult tissues under physiological conditions suggests that the enzyme is a sensible target for drug development. To better understand the role of BCAT1 in cancer, we examined its gene expression at the single cell level (scRNAseq) in specimens obtained from lung, colorectal, breast, and ovarian cancer patients (n=36; >200,000 single cells). In those samples, Bcat1 gene expression was primarily associated with myeloid cells and fibroblasts with the exception of ovarian cancer in which robust Bcat1 presence was also seen in cancer cells. In tumor infiltrating lymphocytes (TILs), enrichment in Bcat1 gene expression was observed in exhausted CD4+ and CD8+ T cells compared to other T cell subtypes; this was independent of the cancer type examined. We speculated that inhibition of BCAT1 in TILs reverses exhaustion and has a therapeutic impact in cancer. In the syngeneic CT26 colon cancer model, combination treatment of a BCAT1 inhibitor (ERG245; ip administration of 5 mg/kg, given at days 7, 8 and 9 after tumor cell inoculation) and an anti-PD1 antibody (ab) (clone RMP1-14; ip administration given at days 7, 11, 14, and 18) led to eradication of established tumors. In the same model, limited treatment of large tumors (average size of ~600 mm3) with ERG245+anti-PD1 ab suppressed tumor growth and increased the frequency of CD8+ T cells expressing Granzyme B and IFNγ by ~50% compared to anti-PD1 monotherapy. Mechanistically, in vitro treatment of newly activated hCD8+ T cells with ERG245 impaired translocation of the lactate transporter MCT1 to the cell surface and increased the levels of intracellular lactate within 30 min of activation leading to upregulation of transcription factor ATF3. ERG245-driven metabolic reprogramming of CD8+ T cells towards an oxidative phenotype (increased OXPHOS) was also observed at 24h of treatment. Withdrawal of ERG245 restored intracellular lactate levels without reversing the metabolic reprogramming of the cells. We propose that these are elements of a mechanism that links temporal inhibition of BCAT1 to the rise of highly energetic CD8+ T cells with increased cytotoxicity and proliferative capacity in vitro and in vivo. Citation Format: Adonia E. Papathanassiu, Francesca Lodi, Hagar Elkafrawy, Michelangelo Certo, Hong Vu, Jeong Hun Ko, Jacques Behmoaras, Claudio Mauro, Diether Lambrechts. BCAT1 as a druggable target in immuno-oncology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3402.

Srivastava PK, van Eyll J, Godard P, Mazzuferi M, Delahaye-Duriez A, Steenwinckel JV, et al. A systems-level framework for drug discovery identifies Csf1R as an anti-epileptic drug target. Nat Commun. 2018;9(1):3561. doi:10.1038/s41467-018-06008-4.The identification of drug targets is highly challenging, particularly for diseases of the brain. To address this problem, we developed and experimentally validated a general computational framework for drug target discovery that combines gene regulatory information with causal reasoning (“Causal Reasoning Analytical Framework for Target discovery”-CRAFT). Using a systems genetics approach and starting from gene expression data from the target tissue, CRAFT provides a predictive framework for identifying cell membrane receptors with a direction-specified influence over disease-related gene expression profiles. As proof of concept, we applied CRAFT to epilepsy and predicted the tyrosine kinase receptor Csf1R as a potential therapeutic target. The predicted effect of Csf1R blockade in attenuating epilepsy seizures was validated in 3 preclinical models of epilepsy. These results highlight CRAFT as a systems-level framework for target discovery and suggest Csf1R blockade as a novel therapeutic strategy in epilepsy. The CRAFT is applicable to disease settings other than epilepsy.

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.

In the quest for identification of genes for complex disorders, the functional annotation of disease-risk variants in the relevant cell type remains a challenge. Lescoat and colleagues1 pose the question ‘ what is a scleroderma macrophage? ’ and in view of current literature (including the study by Moreno-Moral et al 2), argued how the heterogeneous range of activated macrophages can vary between target organs of interest and possibly across different fibrotic diseases. While we share Lescoat’s point of view on the underlying complexity of functional cells in disease, it remains open to debate whether GM-CSF or M-CSF-mediated differentiation conditions of peripheral blood monocytes are sufficient to fully capture the characteristics of these cells in their disease context. As stated by Lescoat et …

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.

Systems genetics stems from systems biology and similarly employs integrative modeling approaches to describe the perturbations and phenotypic effects observed in a complex system. However, in the case of systems genetics the main source of perturbation is naturally occurring genetic variation, which can be analyzed at the systems-level to explain the observed variation in phenotypic traits. In contrast with conventional single-variant association approaches, the success of systems genetics has been in the identification of gene networks and molecular pathways that underlie complex disease. In addition, systems genetics has proven useful in the discovery of master trans-acting genetic regulators of functional networks and pathways, which in many cases revealed unexpected gene targets for disease. Here we detail the central components of a fully integrated systems genetics approach to complex disease, starting from assessment of genetic and gene expression variation, linking DNA sequence variation to mRNA (expression QTL mapping), gene regulatory network analysis and mapping the genetic control of regulatory networks. By summarizing a few illustrative (and successful) examples, we highlight how different data-modeling strategies can be effectively integrated in a systems genetics study.

Systems genetics stems from systems biology and similarly employs integrative modeling approaches to describe the perturbations and phenotypic effects observed in a complex system. However, in the case of systems genetics the main source of perturbation is naturally occurring genetic variation, which can be analyzed at the systems-level to explain the observed variation in phenotypic traits. In contrast with conventional single-variant association approaches, the success of systems genetics has been in the identification of gene networks and molecular pathways that underlie complex disease. In addition, systems genetics has proven useful in the discovery of master trans-acting genetic regulators of functional networks and pathways, which in many cases revealed unexpected gene targets for disease. Here we detail the central components of a fully integrated systems genetics approach to complex disease, starting from assessment of genetic and gene expression variation, linking DNA sequence variation to mRNA (expression QTL mapping), gene regulatory network analysis and mapping the genetic control of regulatory networks. By summarizing a few illustrative (and successful) examples, we highlight how different data-modeling strategies can be effectively integrated in a systems genetics study.

Activation and differentiation of adaptive and innate immune cells require metabolic reprogramming involving diverse metabolic processes. Understanding these processes affords the opportunity to influence and ultimately re-direct the function and fate of these cells. Here, we present data suggesting that exposure of newly activated human CD8+ T cells to a small molecule inhibitor (ERG245; 50-400 μM) of BCAT1 (the cytosolic isoform of the enzyme responsible for the first step in the catabolism of leucine, isoleucine, and valine) resulted in inhibition of T cell proliferation, inhibition of IFNγ and Granzyme B production, and upregulation of PD-1. The antiproliferative effect, as determined by CFSE dilution, required inhibition of BCAT1 during the first 24 hrs of T cell activation and it was reversible upon early withdrawal of the inhibitor. Early withdrawal of ERG245 reversed the observed immunosuppression, ultimately giving rise to CD8+ T cells with higher proliferative capacity and increased cytotoxicity compared to untreated control cells. Exposure of CD8+ T cells to ERG245 resulted in a distinct metabolic phenotype including reduced concentrations of α-ketoglutaric acid (αKG), and lower levels of trimethylation of the lysine 4 (K4) and lysine 27 (K27) sites of histone 3 (H3), which implies that BCAT1 inhibition has epigenetic effects on human CD8+ T cells by modulating αKG-dependent histone demethylases. Similar observations were made with CD8+ T cells, isolated from the spleens of Bcat1 KO mice. In vivo, ERG245 (given at 5 mg/kg ip, bid at days 0, 1 and 2 of treatment), dramatically increased the efficacy of an anti-PD-1 antibody (clone RMP1-14; given at 10 mg/kg at days 0, 4, 7, and 11 of treatment) in the CT26 colon cancer model. Specifically, the combination of ERG245 and anti-PD-1 eradicated established tumors within a week of treatment initiation (cure rate of >80%), whereas the monotherapies (ERG245 or anti-PD-1 alone) did not improve the disease outcome. The data suggest that the temporal exposure of moderately immunogenic tumors to BCAT1 inhibition sensitizes the tumors to checkpoint inhibition. Citation Format: Adonia E. Papathanassiu, Dong-Wook Kim, Kwon-Sik Park, Jeong Hun Ko, Jacques V. Behmoaras. Immunotuning CD8+ T cells with BCAT1 inhibition to increase the efficacy of anti-PD-1 therapy and to eradicate moderately immunogenic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3200.

A novel mechanism in genetically determined P2RX7 levels in macrophages regulates Nlrp3-inflammasome activation and susceptibility to crescentic glomerulonephritis. P2RX7, a mediator of IL-1β and IL-18 processing and release, is a ligand-gated cation channel that is expressed by macrophages. In experimental Crgn, P2RX7 deficiency attenuates renal injury, but the underlying mechanism is unknown. Here, we show that P2RX7 levels and the expression of several genes belonging to the Nlrp3-inflammasome pathway are up-regulated in the macrophages of the WKY rat, a strain uniquely susceptible to macrophage-dependent NTN. Importantly, following P2RX7 activation, WKY BMDMs produce markedly increased levels of active caspase-1, IL-1β, and IL-18 when compared with the NTN-resistant LEW rat BMDMs. P2RX7 and active IL-1β, IL-18, and caspase-1 protein levels were markedly increased in the WKY nephritic glomeruli 4 days following induction of NTN, and the use of a P2RX7 antagonist reduced the levels of secreted active IL-1β. Interestingly, the post-translational control of P2RX7-mediated inflammasome activation is under the genetic regulation of two previously identified Crgn quantitative trait loci in the BMDMs and nephritic glomeruli of the WKY rat. In conclusion, we propose a novel mechanism, whereby genetically determined P2RX7 levels in macrophages regulate Nlrp3-inflammasome activation and susceptibility to Crgn.

Branched-chain aminotransferases (BCAT) are enzymes that initiate the catabolism of branched-chain amino acids (BCAA), such as leucine, thereby providing macromolecule precursors; however, the function of BCATs in macrophages is unknown. Here we show that BCAT1 is the predominant BCAT isoform in human primary macrophages. We identify ERG240 as a leucine analogue that blocks BCAT1 activity. Selective inhibition of BCAT1 activity results in decreased oxygen consumption and glycolysis. This decrease is associated with reduced IRG1 levels and itaconate synthesis, suggesting involvement of BCAA catabolism through the IRG1/itaconate axis within the tricarboxylic acid cycle in activated macrophages. ERG240 suppresses production of IRG1 and itaconate in mice and contributes to a less proinflammatory transcriptome signature. Oral administration of ERG240 reduces the severity of collagen-induced arthritis in mice and crescentic glomerulonephritis in rats, in part by decreasing macrophage infiltration. These results establish a regulatory role for BCAT1 in macrophage function with therapeutic implications for inflammatory conditions., BCATs catabolize leucine and other BCAAs. Here the authors show that BCAA metabolism affects the broken Krebs cycle, reprogramming macrophages to be less proinflammatory, and show that BCAT1 inhibitor ERG240 can treat arthritis in mice and glomerulonephritis in rats.

The recoding of genetic information through RNA editing contributes to proteomic diversity, but the extent and significance of RNA editing in disease is poorly understood. In particular, few studies have investigated the relationship between RNA editing and disease at a genome-wide level. Here, we developed a framework for the genome-wide detection of RNA sites that are differentially edited in disease. Using RNA-sequencing data from 100 hippocampi from mice with epilepsy (pilocarpine–temporal lobe epilepsy model) and 100 healthy control hippocampi, we identified 256 RNA sites (overlapping with 87 genes) that were significantly differentially edited between epileptic cases and controls. The degree of differential RNA editing in epileptic mice correlated with frequency of seizures, and the set of genes differentially RNA-edited between case and control mice were enriched for functional terms highly relevant to epilepsy, including “neuron projection” and “seizures.” Genes with differential RNA editing were preferentially enriched for genes with a genetic association to epilepsy. Indeed, we found that they are significantly enriched for genes that harbor nonsynonymous de novo mutations in patients with epileptic encephalopathy and for common susceptibility variants associated with generalized epilepsy. These analyses reveal a functional convergence between genes that are differentially RNA-edited in acquired symptomatic epilepsy and those that contribute risk for genetic epilepsy. Taken together, our results suggest a potential role for RNA editing in the epileptic hippocampus in the occurrence and severity of epileptic seizures.

Alport syndrome (AS) is a hereditary glomerulopathy caused by a mutation in type IV collagen genes, which disrupts glomerular basement membrane, leading to progressive glomerulosclerosis and end-stage renal failure. There is at present no cure for AS, and cell-based therapies offer promise to improve renal function. In this study, we found that human first trimester fetal chorionic stem cells (CSC) are able to migrate to glomeruli and differentiate down the podocyte lineage in vitro and in vivo. When transplanted into 7-week-old Alport 129Sv-Col4α3(tm1Dec)/J (-/-) mice, a single intraperitoneal injection of CSC significantly lowered blood urea and urine proteinuria levels over the ensuing 2 weeks. In addition, nearly two-thirds of transplanted -/- mice maintained their weight above the 80% welfare threshold, with both males and females weighing more than age-matched nontransplanted -/- mice. This was associated with less renal cortical fibrosis and interstitial inflammation compared to nontransplanted mice as shown by reduction in murine CD4, CD68, and CD45.2 cells. Transplanted CSC homed to glomeruli, where they expressed CR1, VEGFA, SYNAPTOPODIN, CD2AP, and PODOCIN at the RNA level and produced PODOCIN, CD2AP, and COLIVα3 proteins in nontransplanted -/- mice, indicating that CSC have adopted a podocyte phenotype. Together, these data indicate that CSC may be used to delay progression of renal pathology by a combination of anti-inflammatory effects and replacement of the defective resident podocytes.

Background: Fc receptor-mediated macrophage activation is a major cause of glomerular damage in crescentic glomerulonephritis. Results: We investigated the role of a novel rat Fc receptor, Fcgr3-rs, in human and rat macrophage activation. Conclusion: We showed that this receptor prevents the cell signaling of its paralogue (Fcgr3). Significance: These results provide a novel way to inhibit Fc receptor-mediated cell activation in macrophages., Crescentic glomerulonephritis (Crgn) is a complex disease where the initial insult is often the glomerular deposition of antibodies against intrinsic or deposited antigens in the glomerulus. The role of Fc receptors in the induction and progression of Crgn is increasingly recognized, and our previous studies have shown that copy number variation in Fcgr3 partially explains the genetic susceptibility of the Wistar-Kyoto (WKY) rat to nephrotoxic nephritis, a rat model of Crgn. The Fcgr3-related sequence (Fcgr3-rs) is a novel rat-specific Fc receptor with a cytoplasmic domain 6 amino acids longer than its paralogue, Fcgr3. The Fcgr3-rs gene is deleted from the WKY rat genome, and this deletion is associated with enhanced macrophage activity in this strain. Here, we investigated the mechanism by which the deletion of Fcgr3-rs in the WKY strain leads to increased macrophage activation. By lentivirus-mediated gene delivery, we generated stably transduced U937 cells expressing either Fcgr3-rs or Fcgr3. In these cells, which lack endogenous Fcgr3 receptors, we show that Fcgr3-rs interacts with the common Fc-γ chain but that Fc receptor-mediated phagocytosis and signaling are defective. Furthermore, in primary macrophages, expression of Fcgr3-rs inhibits Fc receptor-mediated functions, because WKY bone marrow-derived macrophages transduced with Fcgr3-rs had significantly reduced phagocytic activity. This inhibitory effect on phagocytosis was mediated by the novel cytoplasmic domain of Fcgr3-rs. These results suggest that Fcgr3-rs may act to inhibit Fcgr3-mediated signaling and phagocytosis and could be considered as a novel mechanism in the modulation of Fc receptor-mediated cell activation in autoimmune diseases.

Crescentic glomerulonephritis (CRGN) is a major cause of rapidly progressive renal failure for which the underlying genetic basis is unknown. Wistar-Kyoto (WKY) rats show marked susceptibility to CRGN, whereas Lewis rats are resistant. Glomerular injury and crescent formation are macrophage dependent and mainly explained by seven quantitative trait loci (Crgn1-7). Here, we used microarray analysis in basal and lipopolysaccharide (LPS)-stimulated macrophages to identify genes that reside on pathways predisposing WKY rats to CRGN. We detected 97 novel positional candidates for the uncharacterized Crgn3-7. We identified 10 additional secondary effector genes with profound differences in expression between the two strains (>5-fold change

The extracellular matrix (ECM) plays an important role in vascular tissue structure, maintenance, and function. Lysyl oxidases catalyze a key step in the posttranslational cross-linking of elastin and collagens in the ECM. Gene knockout studies in mice suggested a role for lysyl oxidase-like (LOXL1) in adult elastin synthesis and a role for its isoform, lysyl oxidase (LOX), in the synthesis of both collagens and elastin during development. However, the relative expression of both isoforms as a function of age is not known and was therefore investigated here. LOX and LOXL1 immunohistochemistry and real-time RT-PCR were performed during development, growth and aging in the aorta of LOU and Brown-Norway (BN) rats, two inbred strains with different susceptibilities to arterial fragility. In addition, expression of genes encoding for elastic fiber proteins and type I collagen, together with elastin and collagen contents, was measured in adult and old rat aortas. High aortic LOX expression was observed early in the development (embryonic day 15), followed by a drastic reduction in adulthood, whereas LOXL1 was mainly detectable in the intima and media; its expression was maintained throughout life in the LOU rat. Expression of tropoelastin, type-I collagen, and LOXL1 genes was reduced in the aorta of 6-week-old BN rats. Aging is characterized by a decreased elastin/collagen ratio and a greatly decreased expression of LOX, tropoelastin, and type-I collagen. These findings indicate a different spatial and temporal expression of LOX and LOXL1 during growth and aging in the rat aorta and suggest specific roles for LOX and LOXL1 in the synthesis and remodeling of elastic and collagen fibers.

The Wistar-Kyoto (WKY) rat shows marked susceptibility to crescentic glomerulonephritis. In the model of nephrotoxic nephritis (NTN) that is induced by a small dose of nephrotoxic globulin, WKY rats developed crescents in 80 +/- 2% of glomeruli at day 10, whereas no crescents were seen in Lewis rats. This was associated with marked increase in monocyte chemoattractant protein-1 synthesis in WKY glomeruli. It was posited whether susceptibility depended on circulating cells or intrinsic renal cells. Bone marrow (BM) isografts from WKY to WKY or Lewis to Lewis did not affect susceptibility to NTN. When BM was transferred from WKY to Lewis rats, crescents developed in 35 +/- 9% of glomeruli 10 d after induction of NTN, indicating that susceptibility could be transferred by BM cells. However, crescents were also seen in WKY rats that were given Lewis marrow. For assessment of the contribution of intrinsic renal cells, kidneys from WKY or Lewis rats were transplanted into F1 animals. In NTN, the ratio of crescents in the transplanted kidney to the native kidney was significantly higher for WKY-to-F1 than for Lewis-to-F1 transplants, demonstrating that the kidney itself also influences susceptibility. Mesangial cell responses were then examined in the two strains. Mesangial cells that were derived from WKY rats synthesized significantly more monocyte chemoattractant protein-1 basally and after stimulation with heat-aggregated rabbit IgG or TNF-alpha. These results show that susceptibility to NTN in the WKY rat depends on both circulating and intrinsic renal cells and that there are genetic differences between the strains in mesangial responses to inflammatory stimuli.

Although the effect of genetic background on obesity-related phenotypes is well established, the main objective of this study is to determine the phenotypic responses to cafeteria diet (CAF) of two genetically distinct inbred rat strains and give insight into the molecular mechanisms that might be underlying. Lewis (LEW) and Wistar–Kyoto (WKY) rats were fed with either a standard or a CAF diet. The effects of the diet and the strain in the body weight gain, food intake, respiratory quotient, biochemical parameters in plasma as well as in the expression of genes that regulate leptin signalling were determined. Whereas CAF diet promoted weight gain in LEW and WKY rats, as consequence of increased energy intake, metabolic management of this energy surplus was significantly affected by genetic background. LEW and WKY showed a different metabolic profile, LEW rats showed hyperglycaemia, hypertriglyceridemia and high FFA levels, ketogenesis, high adiposity index and inflammation, but WKY did not. Leptin signalling, and specifically the LepRb-mediated regulation of STAT3 activation and Socs3 gene expression in the hypothalamus were inversely modulated by the CAF diet in LEW (upregulated) and WKY rats (downregulated). In the present study, we show evidence of gene-environment interactions in obesity exerted by differential phenotypic responses to CAF diet between LEW and WKY rats. Specifically, we found the leptin-signalling pathway as a divergent point between the strain-specific adaptations to diet.

Gene-regulatory network analysis is a powerful approach to elucidate the molecular processes and\ud pathways underlying complex disease. Here we employ systems genetics approaches to characterize the\ud genetic regulation of pathophysiological pathways in human temporal lobe epilepsy (TLE). Using\ud surgically acquired hippocampi from 129 TLE patients, we identify a gene-regulatory network genetically\ud associated with epilepsy that contains a specialized, highly expressed transcriptional module encoding\ud proconvulsive cytokines and Toll-like receptor signalling genes. RNA sequencing analysis in a mouse\ud model of TLE using 100 epileptic and 100 control hippocampi shows the proconvulsive module is\ud preserved across-species, specific to the epileptic hippocampus and upregulated in chronic epilepsy. In\ud the TLE patients, we map the trans-acting genetic control of this proconvulsive module to Sestrin 3\ud (SESN3), and demonstrate that SESN3 positively regulates the module in macrophages, microglia and\ud neurons. Morpholino-mediated Sesn3 knockdown in zebrafish confirms the regulation of the\ud transcriptional module, and attenuates chemically induced behavioural seizures in vivo.

Mesangial cells are glomerular cells of stromal origin. During immune complex mediated crescentic glomerulonephritis (Crgn), infiltrating and proliferating pro-inflammatory macrophages lead to crescent formation. Here we have hypothesised that mesangial cells, given their mesenchymal stromal origin, show similar immunomodulatory properties as mesenchymal stem cells (MSCs), by regulating macrophage function associated with glomerular crescent formation. We show that rat mesangial cells suppress conA-stimulated splenocyte proliferation in vitro, as previously shown for MSCs. We then investigated mesangial cell-macrophage interaction by using mesangial cells isolated from nephrotoxic nephritis (NTN)-susceptible Wistar Kyoto (WKY) and NTN-resistant Lewis (LEW) rats. We first determined the mesangial cell transcriptome in WKY and LEW rats and showed that this is under marked genetic control. Supernatant transfer results show that WKY mesangial cells shift bone marrow derived macrophage (BMDM) phenotype to M1 or M2 according to the genetic background (WKY or LEW) of the BMDMs. Interestingly, these effects were different when compared to those of MSCs suggesting that mesangial cells can have unique immunomodulatory effects in the kidney. These results demonstrate the importance of the genetic background in the immunosuppressive effects of cells of stromal origin and specifically of mesangial cell-macrophage interactions in the pathophysiology of crescentic glomerulonephritis.

Summary Crescentic glomerulonephritis (CRGN) is a major cause of human kidney failure, but the underlying mechanisms are not fully understood. Wistar Kyoto (WKY) rats are uniquely susceptible to CRGN following injection of nephrotoxic serum, whereas Lewis (LEW) rats are resistant. Our previous genetic studies of nephrotoxic nephritis (NTN), a form of CRGN induced by nephrotoxic serum, identified Fcgr3 and Jund as WKY genes underlying the two strongest quantitative trait loci for NTN phenotypes: Crgn1 and Crgn2, respectively. We also showed that introgression of WKY Crgn1 or Crgn2 individually into a LEW background did not lead to the formation of glomerular crescents. We have now generated a bicongenic strain, LEW.WCrgn1,2, in which WKY Crgn1 and Crgn2 are both introgressed into the LEW genetic background. These rats show development of NTN phenotypes, including glomerular crescents. Furthermore, we characterised macrophage function and glomerular cytokine profiles in this new strain. Additionally, we show that LEW.WCrgn1,2 rats are resistant to the development of glomerular crescents that is usually induced following immunisation with recombinant rat α3(IV)NC1, the specific Goodpasture autoantigen located in the glomerular basement membrane against which the immune response is directed in experimental autoimmune glomerulonephritis. Our results show that the new bicongenic strain responds differently to two distinct experimental triggers of CRGN. This is the first time that CRGN has been induced on a normally resistant rat genetic background and identifies the LEW.WCrgn1,2 strain as a new, potentially valuable model of macrophage-dependent glomerulonephritis.

The Wistar Kyoto (WKY) rat is uniquely susceptible to experimentally induced crescentic glomerulonephritis. Two major quantitative trait loci (QTLs) on chromosomes 13 (Crgn1) and 16 (Crgn2) with logarithm of odds8, as well as five other loci (Crgn3 through 7), largely explain this genetic susceptibility. To understand further the effects of Crgn1 and Crgn2, we generated a double-congenic strain by introgressing these loci from glomerulonephritis-resistant Lewis rats onto the WKY genetic background. Induction of nephrotoxic nephritis in the double-congenic rats (WKY.LCrgn1,2) produced markedly fewer glomerular crescents, reduced macrophage infiltration, and decreased expression of glomerular TNF-alpha and inducible nitric oxide synthase expression compared with control animals. Bone marrow and kidney transplantation studies between parental and WKY.LCrgn1,2 strains, together with in vitro experiments, demonstrated that Crgn1 and Crgn2 contribute exclusively to circulating cell-related glomerular injury by regulating macrophage infiltration and activation. The residual genetic susceptibility to crescentic glomerulonephritis in WKY.LCrgn1,2 rats associated with macrophage activity (especially with enhanced metalloelastase expression) rather than macrophage infiltration. Taken together, these results demonstrate that a genetic influence on macrophage activation, rather than number, determines glomerular damage in immune-mediated glomerulonephritis.

Genetic mapping and positional cloning of genetically complex traits in the laboratory rat (Rattus norvegicus) has recently led to the identification of various susceptibility genes in different rat models. Rat genetics has benefited from revolutionary advances in molecular biology, genetics, genomics and informatics and provide an unparalleled resource for molecular genetic investigation of mammalian physiopathology and its underlying complex genetic architecture. In this review, we will consider different strategies that are being used in the successful positional cloning of rat complex trait genes in the context of recent progress in rodent and human genetics.

Genetic mapping and positional cloning of genetically complex traits in the laboratory rat (Rattus norvegicus) has recently led to the identification of various susceptibility genes in different rat models. Rat genetics has benefited from revolutionary advances in molecular biology, genetics, genomics and informatics and provide an unparalleled resource for molecular genetic investigation of mammalian physiopathology and its underlying complex genetic architecture. In this review, we will consider different strategies that are being used in the successful positional cloning of rat complex trait genes in the context of recent progress in rodent and human genetics.

La quantite et/ou la qualite des fibres elastiques arterielles sont modifiees dans plusieurs situations pathologiques (anevrisme, atherosclerose,…) et au cours du vieillissement. C’est la raison pour laquelle nous etudions les genes qui regulent la composition de la matrice extracellulaire arterielle et plus particulierement les genes regulateurs de la synthese de l’elastine. Compare a plusieurs autres souches de rat, le rat Brown Norway (BN) a une quantite reduite d’elastine dans son aorte ; ceci est liee a une synthese deficiente de tropoelastine chez le rat en croissance (Sauvage et al, 1997 et 1999 ; Behmoaras et al, 2004). En utilisant des rats F2 BN × LOU, la mesure de la quantite d’elastine dans leur aorte thoracique et l’analyse de 200 marqueurs polymorphiques sur leur ADN, trois “Quantitative Trait Loci (QTL)” controlant la quantite d’elastine arterielle ont ete mis en evidence : deux sur le chromosome 2 et un sur le chromosome 14 (Gauguier et al, 2005). Parallelement, l’utilisation de puces a permis l’analyse des ARNm differentiellement exprimes dans l’aorte des rats BN et des rats LOU de 6 semaines. Ces deux methodes complementaires nous ont permis de mettre en evidence les genes potentiellement regulateurs de la synthese de l’elastine. Plusieurs genes candidats regulent les concentrations de potassium intracellulaire. Nous avons donc teste in vitro et in vivo des agents anti-hypertenseurs ouvreurs de canaux potassiques ATP-dependants tel le minoxidil (Mx) : des CML d’aorte de rat BN ont ete traitees avec le minoxidil sulfate avant de mesurer la quantite d’ARNmE, la transcription du gene elastine et la stabilite des ARNmE ; des rats BN de 3 semaines ont ete traites pendant 10 semaines avec le Mx (22 mg/kg/j) avant d’analyser la quantite d’elastine dans l’aorte. Nous avons ainsi demontre que le Mx stimule la synthese de l’elastine via l’augmentation simultanee de la transcription du gene et de la stabilite des ARNm. Le traitement des rats BN avec Mx induit une augmentation de la quantite d’elastine arterielle. Le minoxidil peut donc etre potentiellement utilisee chez les patients deficients en elastine arterielle.

Crescentic glomerulonephritis is an important cause of human kidney failure for which the underlying molecular basis is largely unknown. In previous studies, we mapped several susceptibility loci, Crgn1-Crgn7, for crescentic glomerulonephritis in the Wistar Kyoto (WKY) rat. Here we show by combined congenic, linkage and microarray studies that the activator protein-1 (AP-1) transcription factor JunD is a major determinant of macrophage activity and is associated with glomerulonephritis susceptibility. Introgression of Crgn2 from the nonsusceptible Lewis strain onto the WKY background leads to significant reductions in crescent formation, macrophage infiltration, Fc receptor-mediated macrophage activation and cytokine production. Haplotype analysis restricted the Crgn2 linkage interval to a 430-kb interval containing Jund, which is markedly overexpressed in WKY macrophages and glomeruli. Jund knockdown in rat and human primary macrophages led to significantly reduced macrophage activity and cytokine secretion, indicating conservation of JunD function in macrophage activation in rats and humans and suggesting in vivo inhibition of Jund as a possible new therapeutic strategy for diseases characterized by inflammation and macrophage activation.

The Brown Norway (BN) rat presents several genetically determined arterial phenotypes of interest, i.e., ruptures of the internal elastic lamina (RIEL) in the abdominal aorta (AA), iliac (IAs), and renal arteries, aortic elastin deficit and higher frequency of persistent ductus arteriosus (PDA) than other strains. We investigated the genetic basis of these phenotypes. We established a backcross between BN and the LOU reference strain and performed a genome-wide scan on 104 males and 105 females with 193 microsatellite markers followed by linkage analysis. RIEL in AA and IAs showed highly significant linkage to a locus on chromosome 5 and suggestive linkage to a locus on chromosome 10, which is syntenic to one linked to a syndrome of thoracic aortic aneurysms with PDA in humans. In contrast, renal artery RIEL mapped to a chromosome 3 locus and thoracic aortic elastic content to two loci on chromosome 2. PDA was significantly linked to two different quantitative trait loci (QTL) on chromosomes 8 and 9. This is the first study in rats to identify genetic loci for PDA. We identified 21 candidate genes by functional relevance or integration of our mapping data with global expression analysis. Sequencing these genes identified 47 single nucleotide polymorphisms, but no functionally relevant amino acid changes. By expression analysis, myosin heavy chain 10, nonmuscle, in the chromosome 10 QTL, emerged as a candidate for RIEL in AA and IAs. Furthermore, production of a congenic line for the chromosome 5 QTL proved implication of this locus in RIEL formation.

Extracellular matrix molecules such as elastin and collagens provide mechanical support to the vessel wall. In addition to its structural role, elastin is a regulator that maintains homeostasis through biologic signaling. Genetically determined minor modifications in elastin and collagen in the aorta could influence the onset and evolution of arterial pathology, such as hypertension and its complications. We previously demonstrated that the inbred Brown Norway (BN) rat shows an aortic elastin deficit in both abdominal and thoracic segments, partly because of a decrease in tropoelastin synthesis when compared with the LOU rat, that elastin gene polymorphisms in these strains do not significantly account for. After a genome-wide search for quantitative trait loci (QTL) influencing the aortic elastin, collagen, and cell protein contents in an F2 population derived from BN and LOU rats, we identified on chromosomes 2 and 14, 3 QTL specifically controlling elastin levels, and a further highly significant QTL on chromosome 17 linked to the level of cell proteins. We also mapped 3 highly significant QTL linked to body weight (on chromosomes 1 and 3) and heart weight (on chromosome 1) in the cross. This study demonstrates the polygenic control of the content of key components of the arterial wall. Such information represents a first step in understanding possible mechanisms involved in dysregulation of these parameters in arterial pathology.