RESUMEN
Despite the recent advances in our understanding of the role of lipids, metabolites, and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving impaired kidney function. The limited availability of kidney biopsies from living donors with acute kidney injury has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study acute kidney injury in humans and characterized these kidneys using imaging and multi-omics approaches. We noted consistent changes in kidney injury and inflammatory markers in donors with reduced kidney function. Neighborhood and correlation analyses of imaging mass cytometry data showed that subsets of kidney cells (proximal tubular cells and fibroblasts) are associated with the expression profile of kidney immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that kidney arachidonic acid metabolism and seven other metabolic pathways were upregulated following diminished kidney function. To validate the arachidonic acid pathway in impaired kidney function we demonstrated increased levels of cytosolic phospholipase A2 protein and related lipid mediators (prostaglandin E2) in the injured kidneys. Further, inhibition of cytosolic phospholipase A2 reduced injury and inflammation in human kidney proximal tubular epithelial cells in vitro. Thus, our study identified cell types and metabolic pathways that may be critical for controlling inflammation associated with impaired kidney function in humans.
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Lesión Renal Aguda , Fenotipo , Humanos , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/etiología , Masculino , Persona de Mediana Edad , Metabolómica/métodos , Femenino , Trasplante de Riñón/efectos adversos , Adulto , Citometría de Imagen/métodos , Riñón/patología , Riñón/metabolismo , Fosfolipasas A2/metabolismo , Ácido Araquidónico/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Transcriptoma , Dinoprostona/metabolismo , Dinoprostona/análisis , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células Epiteliales/metabolismo , Células Epiteliales/patología , Biopsia , MultiómicaRESUMEN
Phytochemicals have a long and successful history in drug discovery. With recent advancements in analytical techniques and methodologies, discovering bioactive leads from natural compounds has become easier. Computational techniques like molecular docking, QSAR modelling and machine learning, and network pharmacology are among the most promising new tools that allow researchers to make predictions concerning natural products' potential targets, thereby guiding experimental validation efforts. Additionally, approaches like LC-MS or LC-NMR speed up compound identification by streamlining analytical processes. Integrating structural and computational biology aids in lead identification, thus providing invaluable information to understand how phytochemicals interact with potential targets in the body. An emerging computational approach is machine learning involving QSAR modelling and deep neural networks that interrelate phytochemical properties with diverse physiological activities such as antimicrobial or anticancer effects.
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Descubrimiento de Drogas , Fitoquímicos , Relación Estructura-Actividad Cuantitativa , Fitoquímicos/química , Fitoquímicos/farmacología , Descubrimiento de Drogas/métodos , Humanos , Aprendizaje Automático , Simulación del Acoplamiento Molecular , Productos Biológicos/química , Productos Biológicos/farmacologíaRESUMEN
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.
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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/patologíaRESUMEN
The GPIbT-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIbαΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbαΔsig/Δsig platelets bound VWF normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a oIIbI3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GpIbαΔsig/Δsig platelets with ADP and thrombin was normal, but GpIbαΔsig/Δsig platelets stimulated with collagen-related-peptide (CRP) exhibited markedly decreased P-selectin exposure and eIIbI3 activation, suggesting a role for the GpIbaaintracellular tail in GPVI-mediated signaling. Consistent with this, while haemostasis was normal in GPIbαΔsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GpIbαΔsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GpIbαΔsig/Δsig platelets but to a lesser extent than those with CRP. GpIbαΔsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of bIIbI3- or GPVI-blockers suggested reduced bIIbI3 activation contributes to the phenotype of the GpIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbiiin transducing both VWF-GPIbGGand collagen-GPVI signaling events in platelets.
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Plaquetas , Factor de von Willebrand , Animales , Plaquetas/metabolismo , Colágeno/metabolismo , Hemostasis , Humanos , Ratones , Transducción de Señal , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismoRESUMEN
Experimental autoimmune vasculitis (EAV) is a model of antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) induced by immunisation of susceptible rat strains with myeloperoxidase (MPO). Animals develop circulating MPO-ANCA, pulmonary haemorrhage, and glomerulonephritis, although renal injury is mild and recovers spontaneously without treatment. In this study we aimed to augment the severity of glomerulonephritis. Following induction of EAV on day 0, a sub-nephritogenic dose of nephrotoxic serum (NTS) containing heterologous antibodies to glomerular basement membrane was administered on day 14. This resulted in a significant increase in disease severity at day 28 compared to MPO immunisation alone - with more urinary abnormalities, infiltrating glomerular leucocytes, and crescent formation that progressed to glomerular and tubulointerstitial scarring by day 56, recapitulating important features of human disease. Importantly, the glomerulonephritis remained pauci-immune, and was strictly dependent on the presence of autoimmunity to MPO, as there was no evidence of renal disease following administration of sub-nephritogenic NTS alone or after immunisation with a control protein in place of MPO. Detailed phenotyping of glomerular leucocytes identified an early infiltrate of non-classical monocytes following NTS administration that, in the presence of autoimmunity to MPO, may initiate the subsequent influx of classical monocytes which augment glomerular injury. We also showed that this model can be used to test novel therapeutics by using a small molecule kinase inhibitor (fostamatinib) that rapidly attenuated both glomerular and pulmonary injury over a 4-day treatment period. We believe that this enhanced model of MPO-AAV will prove useful for the study of glomerular leucocyte behaviour and novel therapeutics in AAV in the future. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
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Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/inmunología , Autoantígenos/inmunología , Modelos Animales de Enfermedad , Glomerulonefritis/inmunología , Peroxidasa/inmunología , Animales , Autoanticuerpos/inmunología , Membrana Basal Glomerular/inmunología , Masculino , RatasRESUMEN
Diabetic kidney disease (DKD) is the leading cause of kidney failure worldwide. Mortality and morbidity associated with DKD are increasing with the global prevalence of type 2 diabetes. Chronic, sub-clinical, non-resolving inflammation contributes to the pathophysiology of renal and cardiovascular disease associated with diabetes. Inflammatory biomarkers correlate with poor renal outcomes and mortality in patients with DKD. Targeting chronic inflammation may therefore offer a route to novel therapeutics for DKD. However, the DKD patient population is highly heterogeneous, with varying etiology, presentation and disease progression. This heterogeneity is a challenge for clinical trials of novel anti-inflammatory therapies. Here, we present a conceptual model of how chronic inflammation affects kidney function in five compartments: immune cell recruitment and activation; filtration; resorption and secretion; extracellular matrix regulation; and perfusion. We believe that the rigorous alignment of pathophysiological insights, appropriate animal models and pathology-specific biomarkers may facilitate a mechanism-based shift from recruiting 'all comers' with DKD to stratification of patients based on the principal compartments of inflammatory disease activity.
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Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Animales , Biomarcadores , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Nefropatías Diabéticas/etiología , Humanos , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , RiñónRESUMEN
BACKGROUND & AIMS: Checkpoint inhibitor-related hepatitis (CPI-Hep) is an emerging clinical challenge. We aimed to gain insights into the immunopathology of CPI-Hep by comprehensively characterising myeloid and lymphoid subsets. METHODS: CPI-treated patients with or without related hepatitis (CPI-Hep; n = 22 and CPI-noHep; n = 7) were recruited. Phenotypic and transcriptional profiling of peripheral immune subsets was performed and compared with 19 healthy controls (HCs). In vitro monocyte-derived macrophages (MoMFs) were assessed for activation and cytokine production. CD163, CCR2, CD68, CD3, CD8 and granzyme B expression was assessed using immunohistochemistry/immunofluorescence (n = 4). RESULTS: A significant total monocyte depletion was observed in CPI-Hep compared with HCs (p = 0.04), along with a proportionate increase in the classical monocyte population (p = 0.0002) and significant upregulation of CCR2, CD163 and downregulation of CCR7. Soluble CD163 levels were significantly elevated in CPI-Hep compared with HCs (p <0.0001). In vitro MoMFs from CPI-Hep showed enhanced production of pro-inflammatory cytokines. CD8+ T cells demonstrated increased perforin, granzyme B, ICOS and HLA-DR expression in CPI-Hep. Transcriptional profiling indicated the presence of activated monocyte and enhanced effector CD8+ T cell populations in CPI-Hep. Immunohistochemistry demonstrated co-localisation of CD8+/granzyme B+ T cells with CD68+CCR2+/CD68+CD163+ macrophages in CPI-Hep liver tissue. CONCLUSIONS: CPI-Hep is associated with activation of peripheral monocytes and an enhanced cytotoxic, effector CD8+ T cell phenotype. These changes were reflected by liver inflammation composed of CD163+/CCR2+ macrophages and CD8+ T cells. LAY SUMMARY: Some patients who receive immunotherapy for cancer develop liver inflammation, which requires cessation of cancer treatment. Herein, we describe ways in which the white blood cells of patients who develop liver inflammation differ from those of patients who receive the same immunotherapy but do not experience liver-related side effects. Targeting some of the pathways we identify may help to prevent or manage this side effect and facilitate cancer treatment.
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Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Antineoplásicos/efectos adversos , Linfocitos T CD8-positivos/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Macrófagos/inmunología , Receptores CCR2/inmunología , Receptores CCR7/inmunología , Receptores de Superficie Celular/inmunología , Antineoplásicos/administración & dosificación , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Descubrimiento de Drogas , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Inhibidores de Puntos de Control Inmunológico/inmunología , Activación de Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Linfocitos T Citotóxicos/inmunologíaRESUMEN
BACKGROUND: Immune complexes within glomerular capillary walls cause crescentic GN (CrGN). Monocytes and macrophages are important in mediating CrGN, but little work has been done to phenotype the subpopulations involved and determine their respective contributions to glomerular inflammation. METHODS: Live glomerular imaging using confocal microscopy monitored intravascular monocyte subset behavior during nephrotoxic nephritis (NTN) in a novel WKY-hCD68-GFP monocyte/macrophage reporter rat strain. Flow cytometry and qPCR further analyzed ex vivo the glomerular leukocyte infiltrate during NTN. RESULTS: Non-classical monocytes surveyed the glomerular endothelium via lymphocyte function-associated antigen 1 (LFA-1) in the steady state. During NTN, non-classical monocytes were recruited first, but subsequent recruitment and retention of classical monocytes was associated with glomerular damage. Monocytes recruited to the glomerular vasculature did not undergo transendothelial migration. This finding suggests that inflammation in immune complex-mediated CrGN is predominantly intravascular, driven by dynamic interactions between intravascular blood monocytes and the endothelium. Glomerular endothelium and non-classical monocytes overexpressed a distinct chemokine axis, which may orchestrate inflammatory myeloid cell recruitment and expression of damage mediators. Reduced classical monocyte recruitment in Lewis rats during NTN confirmed a role for CD16 in mediating glomerular damage. CONCLUSIONS: Monocyte subsets with distinct phenotypes and effector functions may be important in driving inflammation in experimental CrGN resulting from immune complexes formed within the glomerular capillary wall. LFA-1-dependent endothelial surveillance by non-classical monocytes may detect immune complexes through CD16, orchestrating the inflammatory response through intravascular retention of classical monocytes, which results in glomerular damage and proteinuria.
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Endotelio/patología , Glomerulonefritis/inmunología , Glomerulonefritis/patología , Glomérulos Renales/inmunología , Monocitos/patología , Monocitos/fisiología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Capilares , Movimiento Celular , Endotelio/inmunología , Citometría de Flujo , Microscopía Intravital , Glomérulos Renales/diagnóstico por imagen , Masculino , Microscopía Confocal , Monocitos/metabolismo , Fenotipo , Ratas , Receptores de IgG/metabolismoRESUMEN
Monocytes are effectors of the inflammatory response to microbes. Human CD14(+) monocytes specialize in phagocytosis and production of reactive oxygen species and secrete inflammatory cytokines in response to a broad range of microbial cues. Here, we have characterized the functions of human monocytes that lack CD14 (CD14(dim)) and express CD16. CD14(dim) monocytes were genetically distinct from natural killer cells. Gene expression analyses indicated similarities with murine patrolling Gr1(dim) monocytes, and they patrolled the endothelium of blood vessels after adoptive transfer, in a lymphocyte function-associated antigen-1-dependent manner. CD14(dim) monocytes were weak phagocytes and did not produce ROS or cytokines in response to cell-surface Toll-like receptors. Instead, they selectively produced TNF-α, IL-1ß, and CCL3 in response to viruses and immune complexes containing nucleic acids, via a proinflammatory TLR7-TLR 8-MyD88-MEK pathway. Thus, CD14(dim) cells are bona fide monocytes involved in the innate local surveillance of tissues and the pathogenesis of autoimmune diseases.
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Receptores de Lipopolisacáridos/fisiología , Monocitos/fisiología , Ácidos Nucleicos/fisiología , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 8/fisiología , Virus/inmunología , Animales , Presentación de Antígeno , Citocinas/biosíntesis , Proteínas Ligadas a GPI , Antígenos HLA-DR/análisis , Humanos , Lupus Eritematoso Sistémico/inmunología , Ratones , Factor 88 de Diferenciación Mieloide/fisiología , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/análisisRESUMEN
OBJECTIVE: Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. DESIGN: Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer-/-) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. RESULTS: We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer-/- mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. CONCLUSIONS: We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury.
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Fallo Hepático Agudo/inmunología , Fallo Hepático Agudo/metabolismo , Macrófagos/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/farmacología , Tirosina Quinasa c-Mer/metabolismo , Acetaminofén , Adulto , Anciano , Animales , Estudios de Casos y Controles , Femenino , Expresión Génica , Genes MHC Clase II , Antígenos HLA-DR/metabolismo , Humanos , Macrófagos del Hígado/inmunología , Macrófagos del Hígado/metabolismo , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/patología , Macrófagos/inmunología , Masculino , Ratones , Persona de Mediana Edad , Monocitos/inmunología , Monocitos/metabolismo , Neutrófilos/fisiología , Fenotipo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/uso terapéutico , Transcriptoma , Tirosina Quinasa c-Mer/deficiencia , Tirosina Quinasa c-Mer/genéticaRESUMEN
In this chapter, we discuss the manner through which the immune system regulates the cardiovascular system in health and disease. We define the cardiovascular system and elements of atherosclerotic disease, the main focus in this chapter. Herein we elaborate on the disease process that can result in myocardial infarction (heart attack), ischaemic stroke and peripheral arterial disease. We have discussed broadly the homeostatic mechanisms in place that help autoregulate the cardiovascular system including the vital role of cholesterol and lipid clearance as well as the role lipid homeostasis plays in cardiovascular disease in the context of atherosclerosis. We then elaborate on the role played by the immune system in this setting, namely, major players from the innate and adaptive immune system, as well as discussing in greater detail specifically the role played by monocytes and macrophages.This chapter should represent an overview of the role played by the immune system in cardiovascular homeostasis; however further reading of the references cited can expand the reader's knowledge of the detail, and we point readers to many excellent reviews which summarise individual immune systems and their role in cardiovascular disease.
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Arterias/inmunología , Aterosclerosis/inmunología , Sistema Inmunológico/inmunología , Inmunidad Innata , Metabolismo de los Lípidos , Placa Aterosclerótica , Animales , Arterias/metabolismo , Arterias/patología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Comunicación Celular , Humanos , Sistema Inmunológico/metabolismo , Sistema Inmunológico/patología , Factores de Riesgo , Transducción de SeñalRESUMEN
Tissue factor (TF) (CD142) is a 47 kDa transmembrane cell surface glycoprotein that triggers the extrinsic coagulation cascade and links thrombosis with inflammation. Although macrophage TF expression is known to be regulated at the RNA level, very little is known about the mechanisms involved. Poly(adenosine 5'-diphosphate [ADP]-ribose)-polymerase (PARP)-14 belongs to a family of intracellular proteins that generate ADP-ribose posttranslational adducts. Functional screening of PARP-14-deficient macrophages mice revealed that PARP-14 deficiency leads to increased TF expression and functional activity in macrophages after challenge with bacterial lipopolysaccharide. This was related to an increase in TF messenger RNA (mRNA) stability. Ribonucleoprotein complex immunoprecipitation and biotinylated RNA pull-down assays demonstrated that PARP-14 forms a complex with the mRNA-destabilizing protein tristetraprolin (TTP) and a conserved adenylate-uridylate-rich element in the TF mRNA 3' untranslated region. TF mRNA regulation by PARP-14 was selective, as tumor necrosis factor (TNF)α mRNA, which is also regulated by TTP, was not altered in PARP-14 deficient macrophages. Consistent with the in vitro data, TF expression and TF activity, but not TNFα expression, were increased in Parp14(-/-) mice in vivo. Our study provides a novel mechanism for the posttranscriptional regulation of TF expression, indicating that this is selectively regulated by PARP-14.
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Regulación de la Expresión Génica , Macrófagos/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Tromboplastina/biosíntesis , Tristetraprolina/metabolismo , Regiones no Traducidas 3'/fisiología , Animales , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasas/genética , Estabilidad del ARN/efectos de los fármacos , Estabilidad del ARN/fisiología , Tromboplastina/genética , Tristetraprolina/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: The relevance of the dissociation of circulating pentameric C-reactive protein (pCRP) to its monomeric subunits (mCRP) is poorly understood. We investigated the role of conformational C-reactive protein changes in vivo. METHODS AND RESULTS: We identified mCRP in inflamed human striated muscle, human atherosclerotic plaque, and infarcted myocardium (rat and human) and its colocalization with inflammatory cells, which suggests a general causal role of mCRP in inflammation. This was confirmed in rat intravital microscopy of lipopolysaccharide-induced cremasteric muscle inflammation. Intravenous pCRP administration significantly enhanced leukocyte rolling, adhesion, and transmigration via localized dissociation to mCRP in inflamed but not noninflamed cremaster muscle. This was confirmed in a rat model of myocardial infarction. Mechanistically, this process was dependent on exposure of lysophosphatidylcholine on activated cell membranes, which is generated after phospholipase A2 activation. These membrane changes could be visualized intravitally on endothelial cells, as could the colocalized mCRP generation. Blocking of phospholipase A2 abrogated C-reactive protein dissociation and thereby blunted the proinflammatory effects of C-reactive protein. Identifying the dissociation process as a therapeutic target, we stabilized pCRP using 1,6-bis(phosphocholine)-hexane, which prevented dissociation in vitro and in vivo and consequently inhibited the generation and proinflammatory activity of mCRP; notably, it also inhibited mCRP deposition and inflammation in rat myocardial infarction. CONCLUSIONS: These results provide in vivo evidence for a novel mechanism that localizes and aggravates inflammation via phospholipase A2-dependent dissociation of circulating pCRP to mCRP. mCRP is proposed as a pathogenic factor in atherosclerosis and myocardial infarction. Most importantly, the inhibition of pCRP dissociation represents a promising, novel anti-inflammatory therapeutic strategy.
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Proteína C-Reactiva/química , Proteínas Portadoras/química , Inflamación/metabolismo , Músculo Esquelético/metabolismo , Infarto del Miocardio/metabolismo , Miositis/metabolismo , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Biopolímeros , Proteína C-Reactiva/fisiología , Proteínas Portadoras/fisiología , Adhesión Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quimiotaxis de Leucocito , Activación de Complemento , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Hexanos/farmacología , Hexanos/uso terapéutico , Humanos , Inflamación/tratamiento farmacológico , Inflamación/etiología , Rodamiento de Leucocito/efectos de los fármacos , Lipopolisacáridos/toxicidad , Lisofosfatidilcolinas/metabolismo , Masculino , Lípidos de la Membrana/metabolismo , Músculo Esquelético/irrigación sanguínea , Infarto del Miocardio/patología , Miositis/inducido químicamente , Miositis/patología , Inhibidores de Fosfolipasa A2/farmacología , Inhibidores de Fosfolipasa A2/uso terapéutico , Fosfolipasas A2/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Fosforilcolina/uso terapéutico , Estructura Cuaternaria de Proteína , Distribución Aleatoria , Ratas , Ratas Wistar , Receptores de IgG/fisiología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patologíaRESUMEN
OBJECTIVE: Patients with chronic kidney disease (CKD) display impaired cholesterol efflux capacity and elevated CD14(++)CD16(+) monocyte counts. In mice, dysfunctional cholesterol efflux causes monocytosis. It is unknown whether cholesterol efflux capacity and monocyte subsets are associated in CKD. APPROACH AND RESULTS: In 438 patients with CKD, mediators of cholesterol efflux capacity (high-density lipoprotein cholesterol/apolipoprotein A-I) and monocyte subsets were analyzed as predictors of cardiovascular events. Monocyte subset-specific intracellular lipid content, CD36, CD68, and ABCA1 were measured in a subgroup. Experimentally, we analyzed subset-specific cholesterol efflux capacity and response to oxidized low-density lipoprotein cholesterol stimulation in CKD. Epidemiologically, both low Apo-I and low high-density lipoprotein cholesterol were associated with high CD14(++)CD16(+) monocyte counts in linear regression analyses (apolipoprotein A-I: ß=-0.171; P<0.001; high-density lipoprotein cholesterol: ß=-0.138; P=0.005), but not with counts of other monocyte subsets. In contrast to apolipoprotein A-I or high-density lipoprotein cholesterol, higher CD14(++)CD16(+) monocyte counts independently predicted cardiovascular events (hazard ratio per increase of 1 cell/µL: 1.011 [1.003-1.020]; P=0.007). Experimentally, CD14(++)CD16(+) monocytes demonstrated preferential lipid accumulation, high CD36, CD68, and low ABCA1 expression and, consequently, displayed low cholesterol efflux capacity, avid oxidized low-density lipoprotein cholesterol uptake, and potent intracellular interleukin-6, interleukin-1ß, and tumor necrosis factor-α production. CONCLUSIONS: Taken together, mediators of cholesterol efflux are associated with CD14(++)CD16(+) monocyte counts, which independently predict adverse outcome in CKD.
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Apolipoproteína A-I/análisis , Enfermedades Cardiovasculares/epidemiología , HDL-Colesterol/sangre , Monocitos , Insuficiencia Renal Crónica/sangre , Transportador 1 de Casete de Unión a ATP/sangre , Anciano , Enfermedades Cardiovasculares/etiología , Femenino , Proteínas Ligadas a GPI/análisis , Humanos , Inmunofenotipificación , Interleucina-1beta/sangre , Interleucina-6/biosíntesis , Interleucina-6/sangre , Recuento de Leucocitos , Lípidos/sangre , Receptores de Lipopolisacáridos/análisis , Lipoproteínas LDL/sangre , Masculino , Persona de Mediana Edad , Monocitos/clasificación , Estudios Prospectivos , Receptores de IgG/análisis , Insuficiencia Renal Crónica/complicaciones , Método Simple Ciego , Factor de Necrosis Tumoral alfa/análisisRESUMEN
The kidney has a diverse repertoire of cells that make up the mononuclear phagocyte system (MPS). Wu et al. identify a population of CD8αα+CD11c+MHC-II+ blood precursors that display dendritic cell-like characteristics in the glomeruli. These cells show increased recruitment in a rat anti-glomerular basement membrane glomerulonephritis model and were able to attenuate disease. This study highlights the importance of the MPS in kidney disease and the need to better understand it to develop immunotherapeutics translatable to the renal patient.
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Traslado Adoptivo , Autoinmunidad , Antígeno CD11c/sangre , Complejo CD3/sangre , Antígenos CD8/sangre , Linfocitos T CD8-positivos/trasplante , Glomerulonefritis/prevención & control , Antígenos de Histocompatibilidad Clase II/sangre , Glomérulos Renales/inmunología , Animales , FemeninoRESUMEN
Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and selective small-molecule inhibitor of the NLRP3 pathway and has been validated in numerous species and disease models. Although the capacity of MCC950 to block NLRP3 signaling is well-established, it is still critical to identify the mechanism of action and molecular targets of MCC950 to inform and derisk drug development. Quantitative proteomics performed in disease-relevant systems provides a powerful method to study both direct and indirect pharmacological responses to small molecules to elucidate the mechanism of action and confirm target engagement. A comprehensive target deconvolution campaign requires the use of complementary chemical biology techniques. Here we applied two orthogonal chemical biology techniques: compressed Cellular Thermal Shift Assay (CETSA) and photoaffinity labeling chemoproteomics, performed under biologically relevant conditions with LPS-primed THP-1 cells, thereby deconvoluting, for the first time, the molecular targets of MCC950 using chemical biology techniques. In-cell chemoproteomics with inlysate CETSA confirmed the suspected mechanism as the disruption of inflammasome formation via NLRP3. Further cCETSA (c indicates compressed) in live cells mapped the stabilization of NLRP3 inflammasome pathway proteins, highlighting modulation of the targeted pathway. This is the first evidence of direct MCC950 engagement with endogenous NLRP3 in a human macrophage cellular system using discovery proteomics chemical biology techniques, providing critical information for inflammasome studies.
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Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Humanos , Línea Celular , Modelos Animales de Enfermedad , Furanos/farmacología , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteómica , Sulfonamidas/farmacología , Sulfonas/farmacologíaRESUMEN
Introduction: Inflammation is a significant contributor to cardiorenal morbidity and mortality in diabetic kidney disease (DKD). The pathophysiological mechanisms linking systemic, subacute inflammation and local, kidney injury-initiated immune maladaptation is partially understood. Methods: Here, we explored the expression of proinflammatory cytokines in patients with DKD; investigated mouse models of type 1 and type 2 diabetes (T2D); evaluated glomerular signaling in vitro; performed post hoc analyses of systemic and urinary markers of inflammation; and initiated a phase 2b clinical study (FRONTIER-1; NCT04170543). Results: Transcriptomic profiling of kidney biopsies from patients with DKD revealed significant glomerular upregulation of interleukin-33 (IL-33). Inhibition of IL-33 signaling reduced glomerular damage and albuminuria in the uninephrectomized db/db mouse model (T2D/DKD). On a cellular level, inhibiting IL-33 improved glomerular endothelial health by decreasing cellular inflammation and reducing release of proinflammatory cytokines. Therefore, FRONTIER-1 was designed to test the safety and efficacy of the IL-33-targeted monoclonal antibody tozorakimab in patients with DKD. So far, 578 patients are enrolled in FRONTIER-1. The baseline inflammation status of participants (N > 146) was assessed in blood and urine. Comparison to independent reference cohorts (N > 200) validated the distribution of urinary tumor necrosis factor receptor 1 (TNFR1) and C-C motif chemokine ligand 2 (CCL2). Treatment with dapagliflozin for 6 weeks did not alter these biomarkers significantly. Conclusion: We show that blocking the IL-33 pathway may mitigate glomerular endothelial inflammation in DKD. The findings from the FRONTIER-1 study will provide valuable insights into the therapeutic potential of IL-33 inhibition in DKD.
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Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures in vitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages in vitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.
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Eritropoyetina , Células Madre Pluripotentes Inducidas , Humanos , Animales , Ratones , Riñón , Linaje de la Célula , RegeneraciónRESUMEN
BACKGROUND: Checkpoint inhibitor-induced hepatitis (CPI-hepatitis) is an emerging problem with the widening use of CPIs in cancer immunotherapy. Here, we developed a mouse model to characterize the mechanism of CPI-hepatitis and to therapeutically target key pathways driving this pathology. METHODS: C57BL/6 wild-type (WT) mice were dosed with toll-like receptor (TLR)9 agonist (TLR9-L) for hepatic priming combined with anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) plus anti-programmed cell death 1 (PD-1) ("CPI") or phosphate buffered saline (PBS) control for up to 7 days. Flow cytometry, histology/immunofluorescence and messenger RNA sequencing were used to characterize liver myeloid/lymphoid subsets and inflammation. Hepatocyte damage was assessed by plasma alanine transaminase (ALT) and cytokeratin-18 (CK-18) measurements. In vivo investigations of CPI-hepatitis were carried out in Rag2-/- and Ccr2rfp/rfp transgenic mice, as well as following anti-CD4, anti-CD8 or cenicriviroc (CVC; CCR2/CCR5 antagonist) treatment. RESULTS: Co-administration of combination CPIs with TLR9-L induced liver pathology closely resembling human disease, with increased infiltration and clustering of granzyme B+perforin+CD8+ T cells and CCR2+ monocytes, 7 days post treatment. This was accompanied by apoptotic hepatocytes surrounding these clusters and elevated ALT and CK-18 plasma levels. Liver RNA sequencing identified key signaling pathways (JAK-STAT, NF-ΚB) and cytokine/chemokine networks (Ifnγ, Cxcl9, Ccl2/Ccr2) as drivers of CPI-hepatitis. Using this model, we show that CD8+ T cells mediate hepatocyte damage in experimental CPI-hepatitis. However, their liver recruitment, clustering, and cytotoxic activity is dependent on the presence of CCR2+ monocytes. The absence of hepatic monocyte recruitment in Ccr2rfp/rfp mice and CCR2 inhibition by CVC treatment in WT mice was able to prevent the development and reverse established experimental CPI-hepatitis. CONCLUSION: This newly established mouse model provides a platform for in vivo mechanistic studies of CPI-hepatitis. Using this model, we demonstrate the central role of liver infiltrating CCR2+ monocyte interaction with tissue-destructive CD8+ T cells in the pathogenesis of CPI-hepatitis and highlight CCR2 inhibition as a novel therapeutic target.