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1.
EMBO Rep ; 24(3): e56310, 2023 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-36597777

RESUMEN

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.


Asunto(s)
Macrófagos , Osteoclastos , Humanos , Macrófagos/metabolismo , Osteoclastos/metabolismo , Huesos , Células Gigantes , Antígenos B7/metabolismo
2.
Int J Mol Sci ; 25(7)2024 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-38612639

RESUMEN

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful technique for investigating biological heterogeneity at the single-cell level in human systems and model organisms. Recent advances in scRNA-seq have enabled the pooling of cells from multiple samples into single libraries, thereby increasing sample throughput while reducing technical batch effects, library preparation time, and the overall cost. However, a comparative analysis of scRNA-seq methods with and without sample multiplexing is lacking. In this study, we benchmarked methods from two representative platforms: Parse Biosciences (Parse; with sample multiplexing) and 10x Genomics (10x; without sample multiplexing). By using peripheral blood mononuclear cells (PBMCs) obtained from two healthy individuals, we demonstrate that demultiplexed scRNA-seq data obtained from Parse showed similar cell type frequencies compared to 10x data where samples were not multiplexed. Despite relatively lower cell capture affecting library preparation, Parse can detect rare cell types (e.g., plasmablasts and dendritic cells) which is likely due to its relatively higher sensitivity in gene detection. Moreover, a comparative analysis of transcript quantification between the two platforms revealed platform-specific distributions of gene length and GC content. These results offer guidance for researchers in designing high-throughput scRNA-seq studies.


Asunto(s)
Benchmarking , Leucocitos Mononucleares , Humanos , Biblioteca de Genes , Genómica , Análisis de Secuencia de ARN
3.
J Pathol ; 257(3): 300-313, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35239186

RESUMEN

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.


Asunto(s)
Glomerulonefritis , Receptores Purinérgicos P2X7 , Vasculitis , Adenosina Trifosfato/metabolismo , Animales , Caspasa 1/metabolismo , Caspasas , Inflamasomas/metabolismo , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ratas , Ratas Endogámicas WKY , Receptores Purinérgicos P2X7/metabolismo , Vasculitis/metabolismo , Vasculitis/patología
4.
J Cell Sci ; 133(22)2020 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-33148611

RESUMEN

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.


Asunto(s)
Activación de Macrófagos , Macrófagos , Mitocondrias , Transaminasas , Ciclo del Ácido Cítrico , Humanos , Leucina/metabolismo , Macrófagos/metabolismo , Mitocondrias/metabolismo , Transaminasas/metabolismo
5.
BMC Biol ; 19(1): 246, 2021 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-34794433

RESUMEN

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.


Asunto(s)
Células Gigantes , Macrófagos , Fusión Celular , Humanos , Lípidos , Glicoproteínas de Membrana , Osteoclastos , Receptores Inmunológicos
6.
J Cell Sci ; 131(11)2018 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-29871956

RESUMEN

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.


Asunto(s)
Células Gigantes/metabolismo , Macrófagos/metabolismo , Osteoclastos/metabolismo , Transducción de Señal , Animales , Fusión Celular , Granuloma , Humanos
7.
Genome Res ; 27(3): 440-450, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28250018

RESUMEN

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.


Asunto(s)
Epilepsia/genética , Edición de ARN , Animales , Estudio de Asociación del Genoma Completo , Hipocampo/metabolismo , Masculino , Ratones , Transcriptoma
10.
Ann Rheum Dis ; 77(4): 596-601, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29348297

RESUMEN

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.


Asunto(s)
Predisposición Genética a la Enfermedad , Macrófagos/citología , Proteínas de Neoplasias/genética , Esclerodermia Sistémica/genética , Transcriptoma/genética , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Técnicas de Genotipaje , Humanos , Masculino , Sitios de Carácter Cuantitativo/genética , Factores de Riesgo , Esclerodermia Sistémica/patología , Transducción de Señal/genética , Piel/metabolismo , Adulto Joven
11.
J Immunol ; 194(10): 4705-4716, 2015 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-25840911

RESUMEN

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.


Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Fibrosis/enzimología , Fibrosis/genética , Macrófagos/enzimología , Animales , Western Blotting , Cromatografía Liquida , Citocromo P-450 CYP2J2 , Familia 2 del Citocromo P450 , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Técnicas de Inactivación de Genes , Glomerulonefritis/enzimología , Glomerulonefritis/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Interferencia de ARN , Ratas , Ratas Endogámicas WKY , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en Tándem , Transcriptoma
12.
Mol Cell Proteomics ; 14(3): 484-98, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25532521

RESUMEN

Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics (LC-MS/MS) and transcriptome (high-throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift toward HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases. We use shortest-path analysis to link differential phosphorylation with the transcriptomic reprogramming of macrophages and identify LRRFIP1, SMARCA4, and DNMT1 as novel regulators of MM. We experimentally validate these predictions by showing that knock-down of these latter reduce macrophage multinucleation. These results provide a new framework for the combined analysis of transcriptional and post-translational changes during macrophage multinucleation, prioritizing essential genes, and revealing the sequential events leading to the multinucleation of macrophages.


Asunto(s)
Núcleo Celular/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Helicasas/metabolismo , Perfilación de la Expresión Génica/métodos , Macrófagos/metabolismo , Proteínas Nucleares/metabolismo , Proteoma/análisis , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Helicasas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas Nucleares/genética , Fosforilación , Proteínas de Unión al ARN/genética , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WKY , Análisis de Secuencia de ARN/métodos , Factores de Transcripción/genética
14.
Cell Rep ; 43(8): 114516, 2024 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-39024103

RESUMEN

Despite its significance, the role of lipid metabolism in NLRP3 inflammasome remains elusive. Here, we reveal a critical role for fatty acid synthase (FASN) in NLRP3 inflammasome activation. We demonstrate that pharmacological or genetic depletion of FASN dampens NLRP3 activation in primary mouse and human macrophages and in mice. This disruption in NLRP3 activation is contingent upon FASN activity. Accordingly, abolishing cellular palmitoylation, a post-translational modification in which the FASN product palmitate is reversibly conjugated to cysteine residues of target proteins, blunts inflammasome signaling. Correspondingly, an acyl-biotin exchange assay corroborated NLRP3 palmitoylation. Mechanistically, Toll-like receptor (TLR) ligation introduces palmitoylation at NLRP3 Cys898, permitting NLRP3 translocation to dispersed trans-Golgi network (dTGN) vesicles, the site of inflammasome assembly, upon NLRP3 activation. Accordingly, the NLRP3 Cys898 mutant exhibits reduced palmitoylation, limited translocation to the dTGN compartment, and diminished inflammasome activation. These results underscore mechanistic insights through which lipid metabolism licenses NLRP3 inflammasome assembly and activation.


Asunto(s)
Inflamasomas , Lipoilación , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Humanos , Ratones , Acido Graso Sintasa Tipo I/metabolismo , Acido Graso Sintasa Tipo I/genética , Ácidos Grasos/metabolismo , Inflamasomas/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transporte de Proteínas/efectos de los fármacos , Red trans-Golgi/metabolismo
15.
EBioMedicine ; 103: 105127, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38677183

RESUMEN

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.


Asunto(s)
Tejido Adiposo Blanco , Hígado Graso , Obesidad , Oxilipinas , Humanos , Obesidad/metabolismo , Obesidad/complicaciones , Femenino , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado Graso/etiología , Masculino , Oxilipinas/metabolismo , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Persona de Mediana Edad , Adulto , Inflamación/metabolismo , Inflamación/patología , Hígado/metabolismo , Hígado/patología , Biomarcadores , Espectrometría de Masas en Tándem
16.
J Biol Chem ; 287(8): 5710-9, 2012 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-22184119

RESUMEN

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.


Asunto(s)
Glomerulonefritis/metabolismo , Macrófagos/metabolismo , Receptores de IgG/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular Tumoral , Citoplasma/metabolismo , Exones/genética , Eliminación de Gen , Duplicación de Gen , Glomerulonefritis/patología , Humanos , Interleucina-1beta/metabolismo , Macrófagos/patología , Ratones , Datos de Secuencia Molecular , Fagocitosis , Filogenia , Estructura Terciaria de Proteína , Ratas , Receptores de IgG/química , Receptores de IgG/genética , Transducción de Señal , Especificidad de la Especie , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
17.
BMC Genomics ; 14: 92, 2013 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-23398888

RESUMEN

BACKGROUND: The oxidative burst is one of the major antimicrobial mechanisms adopted by macrophages. The WKY rat strain is uniquely susceptible to experimentally induced macrophage-dependent crescentic glomerulonephritis (Crgn). We previously identified the AP-1 transcription factor JunD as a determinant of macrophage activation in WKY bone marrow-derived macrophages (BMDMs). JunD is over-expressed in WKY BMDMs and its silencing reduces Fc receptor-mediated oxidative burst in these cells. RESULTS: Here we combined Jund RNA interference with microarray analyses alongside ChIP-sequencing (ChIP-Seq) analyses in WKY BMDMs to investigate JunD-mediated control of macrophage activation in basal and lipopolysaccharide (LPS) stimulated cells. Microarray analysis following Jund silencing showed that Jund activates and represses gene expression with marked differential expression (>3 fold) for genes linked with oxidative stress and IL-1ß expression. These results were complemented by comparing whole genome expression in WKY BMDMs with Jund congenic strain (WKY.LCrgn2) BMDMs which express lower levels of JunD. ChIP-Seq analyses demonstrated that the increased expression of JunD resulted in an increased number of binding events in WKY BMDMs compared to WKY.LCrgn2 BMDMs. Combined ChIP-Seq and microarray analysis revealed a set of primary JunD-targets through which JunD exerts its effect on oxidative stress and IL-1ß synthesis in basal and LPS-stimulated macrophages. CONCLUSIONS: These findings demonstrate how genetically determined levels of a transcription factor affect its binding sites in primary cells and identify JunD as a key regulator of oxidative stress and IL-1ß synthesis in primary macrophages, which may play a role in susceptibility to Crgn.


Asunto(s)
Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Estrés Oxidativo/genética , Proteínas Proto-Oncogénicas c-jun , Factor de Transcripción AP-1 , Animales , Sitios de Unión , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Glomerulonefritis/inducido químicamente , Glomerulonefritis/genética , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Lipopolisacáridos/toxicidad , Macrófagos/citología , Macrófagos/efectos de los fármacos , Cultivo Primario de Células , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo
18.
Genome Res ; 20(6): 791-803, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20430781

RESUMEN

The spontaneously hypertensive rat (SHR) is the most widely studied animal model of hypertension. Scores of SHR quantitative loci (QTLs) have been mapped for hypertension and other phenotypes. We have sequenced the SHR/OlaIpcv genome at 10.7-fold coverage by paired-end sequencing on the Illumina platform. We identified 3.6 million high-quality single nucleotide polymorphisms (SNPs) between the SHR/OlaIpcv and Brown Norway (BN) reference genome, with a high rate of validation (sensitivity 96.3%-98.0% and specificity 99%-100%). We also identified 343,243 short indels between the SHR/OlaIpcv and reference genomes. These SNPs and indels resulted in 161 gain or loss of stop codons and 629 frameshifts compared with the BN reference sequence. We also identified 13,438 larger deletions that result in complete or partial absence of 107 genes in the SHR/OlaIpcv genome compared with the BN reference and 588 copy number variants (CNVs) that overlap with the gene regions of 688 genes. Genomic regions containing genes whose expression had been previously mapped as cis-regulated expression quantitative trait loci (eQTLs) were significantly enriched with SNPs, short indels, and larger deletions, suggesting that some of these variants have functional effects on gene expression. Genes that were affected by major alterations in their coding sequence were highly enriched for genes related to ion transport, transport, and plasma membrane localization, providing insights into the likely molecular and cellular basis of hypertension and other phenotypes specific to the SHR strain. This near complete catalog of genomic differences between two extensively studied rat strains provides the starting point for complete elucidation, at the molecular level, of the physiological and pathophysiological phenotypic differences between individuals from these strains.


Asunto(s)
Hipertensión/genética , Animales , Codón de Terminación , Dosificación de Gen , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Ratas , Ratas Endogámicas SHR , Transcripción Genética
19.
Am J Pathol ; 180(5): 1843-51, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22445570

RESUMEN

In experimental autoimmune glomerulonephritis (EAG), a model of Goodpasture's disease, Wistar Kyoto (WKY) rats immunized with collagenase-solubilized glomerular basement membrane (GBM) or the recombinant NC1 domain of the α3 chain of type IV collagen [α3(IV)NC1] develop anti-GBM antibodies and focal necrotizing glomerulonephritis with crescent formation. However, Lewis (LEW) rats, which share the same major histocompatibility complex (MHC) haplotype, are resistant to EAG development. A genome-wide linkage analysis of backcrossed animals with EAG revealed a major quantitative trait locus (QTL) on rat chromosome 13 (LOD = 3.9) linked to the percentage of glomerular crescents. To investigate the role of this QTL in EAG induction, reciprocal congenic rats were generated (LEW.WCrgn1 congenic and WKY.LCrgn1 congenic), immunized with recombinant rat α3(IV)NC1, and assessed for EAG development. WKY.LCrgn1 rats showed a marked reduction in albuminuria, severity of crescentic nephritis, and number of glomerular macrophages compared with WKY controls. No reduction in antibody levels was observed. However, LEW.WCrgn1 rats were resistant to EAG development, as were LEW controls. Macrophage activation in vitro was assessed in parental and congenic rat bone marrow-derived macrophages (BMDMs). WKY.LCrgn1 BMDMs showed a significant reduction in Fc receptor-mediated oxidative burst, phagocytosis of opsonised polystyrene beads, and LPS-induced levels of MCP-1 secretion and iNOS mRNA expression compared with WKY rats. These results confirm the importance of Crgn1 on chromosome 13 in EAG susceptibility, mediated partly through differences in Fc receptor-mediated macrophage activation.


Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/genética , Animales , Animales Congénicos , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/patología , Autoanticuerpos/biosíntesis , Autoantígenos/inmunología , Quimiocina CCL2/biosíntesis , Colágeno Tipo IV/inmunología , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Membrana Basal Glomerular/inmunología , Glomérulos Renales/patología , Activación de Macrófagos/genética , Macrófagos/patología , Masculino , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fagocitosis/genética , Fagocitosis/inmunología , Fenotipo , Sitios de Carácter Cuantitativo/genética , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WKY , Receptores Fc/inmunología , Proteínas Recombinantes/inmunología , Estallido Respiratorio/genética , Estallido Respiratorio/inmunología , Especificidad de la Especie
20.
Elife ; 122023 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-37706477

RESUMEN

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.


Asunto(s)
Pulmón , Macrófagos , Femenino , Humanos , Animales , Ratones , Macrófagos/metabolismo , Fibrosis , Pulmón/metabolismo , Matriz Extracelular , Diferenciación Celular
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