RESUMEN
Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a-/-) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFß signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a-/- animals. Treatment of podocytes in vitro with TGF-ß1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-ß1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , MicroARNs/metabolismo , Podocitos/metabolismo , Receptor ErbB-4/biosíntesis , Receptor Notch1/biosíntesis , Regulación hacia Arriba , Animales , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Clorhidrato de Erlotinib/farmacología , Ratones , Ratones Noqueados , MicroARNs/genética , Podocitos/patología , Receptor ErbB-4/genética , Receptor Notch1/genética , Ribonucleasas/genética , Ribonucleasas/metabolismo , Factores de Riesgo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Gain-of-function mutations of classic transient receptor potential channel 6 (TRPC6) were identified in familial FSGS, and increased expression of wild-type TRPC6 in glomeruli is observed in several human acquired proteinuric diseases. Synaptopodin, an actin binding protein that is important in maintaining podocyte function, is downregulated in various glomerular diseases. Here, we investigated whether synaptopodin maintains podocyte function by regulating podocyte surface expression and activity of TRPC6. We show indirect interaction and nonrandom association of synaptopodin and TRPC6 in podocytes. Knockdown of synaptopodin in cultured mouse podocytes increased the expression of TRPC6 at the plasma membrane, whereas overexpression of synaptopodin decreased it. Mechanistically, synaptopodin-dependent TRPC6 surface expression required functional actin and microtubule cytoskeletons. Overexpression of wild-type or FSGS-inducing mutant TRPC6 in synaptopodin-depleted podocytes enhanced TRPC6-mediated calcium influx and induced apoptosis. In vivo, knockdown of synaptopodin also caused increased podocyte surface expression of TRPC6. Administration of cyclosporin A, which stabilizes synaptopodin, reduced LPS-induced proteinuria significantly in wild-type mice but to a lesser extent in TRPC6 knockout mice. Furthermore, administration of cyclosporin A reversed the LPS-induced increase in podocyte surface expression of TRPC6 in wild-type mice. Our findings suggest that alteration in synaptopodin levels under disease conditions may modify intracellular TRPC6 channel localization and activity, which further contribute to podocyte dysfunction. Reducing TRPC6 surface levels may be a new approach to restoring podocyte function.
Asunto(s)
Proteínas de Microfilamentos/fisiología , Podocitos/metabolismo , Proteinuria/metabolismo , Canales Catiónicos TRPC/biosíntesis , Animales , Membrana Celular/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Podocitos/ultraestructura , Canal Catiónico TRPC6RESUMEN
Abatacept (cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin fusion protein [CTLA-4-Ig]) is a costimulatory inhibitor that targets B7-1 (CD80). The present report describes five patients who had focal segmental glomerulosclerosis (FSGS) (four with recurrent FSGS after transplantation and one with primary FSGS) and proteinuria with B7-1 immunostaining of podocytes in kidney-biopsy specimens. Abatacept induced partial or complete remissions of proteinuria in these patients, suggesting that B7-1 may be a useful biomarker for the treatment of some glomerulopathies. Our data indicate that abatacept may stabilize ß1-integrin activation in podocytes and reduce proteinuria in patients with B7-1-positive glomerular disease.
Asunto(s)
Antígeno B7-1/metabolismo , Glomeruloesclerosis Focal y Segmentaria/tratamiento farmacológico , Inmunoconjugados/farmacología , Abatacept , Adolescente , Adulto , Antígeno B7-1/antagonistas & inhibidores , Biomarcadores/metabolismo , Niño , Femenino , Glomeruloesclerosis Focal y Segmentaria/complicaciones , Glomeruloesclerosis Focal y Segmentaria/inmunología , Humanos , Inmunoconjugados/uso terapéutico , Masculino , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Proteinuria/tratamiento farmacológico , Proteinuria/etiología , Adulto JovenRESUMEN
Podocyte injury and loss mark an early step in the pathogenesis of various glomerular diseases, making these cells excellent targets for therapeutics. However, cell-based high-throughput screening assays for the rational development of podocyte-directed therapeutics are currently lacking. Here, we describe a novel high-content screening-based phenotypic assay that analyzes thousands of podocytes per assay condition in 96-well plates to quantitatively measure dose-dependent changes in multiple cellular features. Our assay consistently produced a Z' value >0.44, making it suitable for compound screening. On screening with >2100 pharmacologically active agents, we identified 24 small molecules that protected podocytes against injury in vitro (1% hit rate). Among the identified hits, we confirmed an ß1-integrin agonist, pyrintegrin, as a podocyte-protective agent. Treatment with pyrintegrin prevented damage-induced decreases in F-actin stress fibers, focal adhesions, and active ß1-integrin levels in cultured cells. In vivo, administration of pyrintegrin protected mice from LPS-induced podocyte foot process effacement and proteinuria. Analysis of the murine glomeruli showed that LPS administration reduced the levels of active ß1 integrin in the podocytes, which was prevented by cotreatment with pyrintegrin. In rats, pyrintegrin reduced peak proteinuria caused by puromycin aminonucleoside-induced nephropathy. Our findings identify pyrintegrin as a potential therapeutic candidate and show the use of podocyte-based screening assays for identifying novel therapeutics for proteinuric kidney diseases.
Asunto(s)
Hidroxiquinolinas/química , Integrina beta1/metabolismo , Glomérulos Renales/metabolismo , Podocitos/citología , Sulfonamidas/química , Actinas/metabolismo , Albuminuria/metabolismo , Animales , Movimiento Celular , Células Epiteliales/efectos de los fármacos , Adhesiones Focales/metabolismo , Ensayos Analíticos de Alto Rendimiento , Enfermedades Renales/metabolismo , Lipopolisacáridos/química , Ratones , Microscopía Confocal , Fenotipo , Proteinuria/patología , Puromicina Aminonucleósido/química , RatasRESUMEN
Lung inflammation plays a key role in the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants. The challenge in BPD management is the lack of effective and safe antiinflammatory agents. Leukadherin-1 (LA1) is a novel agonist of the leukocyte surface integrin CD11b/CD18 that enhances leukocyte adhesion to ligands and vascular endothelium and thus reduces leukocyte transendothelial migration and influx to the injury sites. Its functional significance in preventing hyperoxia-induced neonatal lung injury is unknown. We tested the hypothesis that administration of LA1 is beneficial in preventing hyperoxia-induced neonatal lung injury, an experimental model of BPD. Newborn rats were exposed to normoxia (21% O2) or hyperoxia (85% O2) and received twice-daily intraperitoneal injection of LA1 or placebo for 14 days. Hyperoxia exposure in the presence of the placebo resulted in a drastic increase in the influx of neutrophils and macrophages into the alveolar airspaces. This increased leukocyte influx was accompanied by decreased alveolarization and angiogenesis and increased pulmonary vascular remodeling and pulmonary hypertension (PH), the pathological hallmarks of BPD. However, administration of LA1 decreased macrophage infiltration in the lungs during hyperoxia. Furthermore, treatment with LA1 improved alveolarization and angiogenesis and decreased pulmonary vascular remodeling and PH. These data indicate that leukocyte recruitment plays an important role in the experimental model of BPD induced by hyperoxia. Targeting leukocyte trafficking using LA1, an integrin agonist, is beneficial in preventing lung inflammation and protecting alveolar and vascular structures during hyperoxia. Thus, targeting integrin-mediated leukocyte recruitment and inflammation may provide a novel strategy in preventing and treating BPD in preterm infants.
Asunto(s)
Antiinflamatorios/farmacología , Benzoatos/farmacología , Displasia Broncopulmonar/prevención & control , Hiperoxia/tratamiento farmacológico , Tiohidantoínas/farmacología , Animales , Animales Recién Nacidos , Antiinflamatorios/uso terapéutico , Benzoatos/uso terapéutico , Displasia Broncopulmonar/etiología , Evaluación Preclínica de Medicamentos , Hiperoxia/complicaciones , Hipertensión Pulmonar/tratamiento farmacológico , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/inmunología , Infiltración Neutrófila , Ratas Sprague-Dawley , Tiohidantoínas/uso terapéutico , Resultado del Tratamiento , Remodelación VascularRESUMEN
Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pHi). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pHi.
Asunto(s)
Podocitos/metabolismo , Proteinuria/metabolismo , Animales , Transporte Biológico Activo/genética , Transporte Biológico Activo/inmunología , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/inmunología , Citoesqueleto/metabolismo , Citoesqueleto/patología , Concentración de Iones de Hidrógeno , Ratones , Ratones Noqueados , Podocitos/inmunología , Podocitos/patología , Proteinuria/genética , Proteinuria/inmunología , Proteinuria/patologíaRESUMEN
Toll-like receptor (TLR) signaling is an important component in the inflammatory response generated in diseases characterized by autoantibody reactivity to proteins such as SSA/Ro in complex with endogenous nucleic acids. Complement receptor 3 (CR3), a genetic variant of which has been identified as a risk factor in systemic lupus erythematosus, has been shown to induce tolerogenic responses in dendritic cells and suppress TLR4 responses in a murine sepsis model. Accordingly, this study addressed the hypothesis that activation of CR3, influenced by genotype of CD11b, negatively regulates TLR7/8-dependent effector function. Allosteric activation of CD11b via pretreatment with the small molecule, leukadhedrin 1 (LA1), significantly attenuated TLR7/8-induced (hY3 RNA, R848) secretion of TNFα in THP-1 cells and human macrophages isolated from donors homozygous for the ancestral common ITGAM allele at rs1143679. This inhibition was accompanied by profound degradation of the adaptor protein MyD88, an effect not observed with direct inhibition of TLR ligation by an antagonist oligonucleotide. In contrast, the addition of LA1 after incubation with the TLR agonists did not result in MyD88 degradation and subsequent attenuation of TNFα secretion. In TLR7/8-stimulated macrophages isolated from donors heterozygous for the CD11b variant, pretreatment with LA1 did not down-regulate TNFα release. These novel findings support a negative cross-talk between CR3 and TLR pathways likely to be induced by antibodies reactive with ribonucleoproteins and point to the development of CR3-specific agonists as potential therapeutics for diseases such as neonatal lupus.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , Antígeno CD11b/biosíntesis , Regulación de la Expresión Génica , Antígeno de Macrófago-1/metabolismo , Macrófagos/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Sitio Alostérico , Antiinflamatorios/farmacología , Adhesión Celular , Regulación hacia Abajo , Heterocigoto , Humanos , Inflamación , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Macrófagos/citología , Modelos Genéticos , Factor 88 de Diferenciación Mieloide/metabolismo , ARN/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: CD11b/CD18 is a key adhesion receptor that mediates leukocyte adhesion, migration and immune functions. We recently identified novel compounds, leukadherins, that allosterically enhance CD11b/CD18-dependent cell adhesion and reduce inflammation in vivo, suggesting integrin activation to be a novel mechanism of action for the development of anti-inflammatory therapeutics. Since a number of well-characterized anti-CD11b/CD18 activating antibodies are currently available, we wondered if such biological agonists could also become therapeutic leads following this mechanism of action. METHODS: We compared the two types of agonists using in vitro cell adhesion and wound-healing assays and using animal model systems. We also studied effects of the two types of agonists on outside-in signaling in treated cells. RESULTS: Both types of agonists similarly enhanced integrin-mediated cell adhesion and decreased cell migration. However, unlike leukadherins, the activating antibodies produced significant CD11b/CD18 macro clustering and induced phosphorylation of key proteins involved in outside-in signaling. Studies using conformation reporter antibodies showed that leukadherins did not induce global conformational changes in CD11b/CD18 explaining the reason behind their lack of ligand-mimetic outside-in signaling. In vivo, leukadherins reduced vascular injury in a dose-dependent fashion, but, surprisingly, the anti-CD11b activating antibody ED7 was ineffective. CONCLUSIONS: Our results suggest that small molecule allosteric agonists of CD11b/CD18 have clear advantages over the biologic activating antibodies and provide a mechanistic basis for the difference. GENERAL SIGNIFICANCE: CD11b/CD18 activation represents a novel strategy for reducing inflammatory injury. Our study establishes small molecule leukadherins as preferred agonists over activating antibodies for future development as novel anti-inflammatory therapeutics.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Anticuerpos Monoclonales de Origen Murino/farmacología , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Arteria Ilíaca/lesiones , Transducción de Señal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Antiinflamatorios no Esteroideos/química , Antígeno CD11b/genética , Antígenos CD18/genética , Adhesión Celular/efectos de los fármacos , Humanos , Arteria Ilíaca/metabolismo , Arteria Ilíaca/patología , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Células K562 , Ratones , Estructura Cuaternaria de Proteína , Ratas , Ratas Sprague-DawleyRESUMEN
Aging has been associated with pathological vascular remodeling and increased neointimal hyperplasia. The understanding of how aging exacerbates this process is fundamental to prevent cardiovascular complications in the elderly. This study proposes a mechanism by which aging sustains leukocyte adhesion, vascular inflammation, and increased neointimal thickness after injury. The effect of aging on vascular remodeling was assessed in the rat balloon injury model using microarray analysis, immunohistochemistry, and LINCOplex assays. The injured arteries in aging rats developed thicker neointimas than those in younger animals, and this significantly correlated with a higher number of tissue macrophages and increased vascular IL-18. Indeed, IL-18 was 23-fold more abundant in the injured vasculature of aged animals compared with young rats, while circulating levels were similar in both groups of animals. The depletion of macrophages in aged rats with clodronate liposomes ameliorated vascular accumulation of IL-18 and significantly decreased neointimal formation. IL-18 was found to inhibit apoptosis of vascular smooth muscle cells (VSMC) and macrophages, thus favoring both the formation and inflammation of the neointima. In addition, injured arteries of aged rats accumulated 18-fold more fibrinogen-γ than those of young animals. Incubation of rat peritoneal macrophages with immobilized IL-18 increased leukocyte adhesion to fibrinogen and suggested a proinflammatory positive feedback loop among macrophages, VSMC, and the deposition of fibrinogen during neointimal hyperplasia. In conclusion, our data reveal that concentration changes in vascular cytokine and fibrinogen following injury in aging rats contribute to local inflammation and postinjury neointima formation.
Asunto(s)
Envejecimiento/metabolismo , Fibrinógeno/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-18/metabolismo , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Neointima , Comunicación Paracrina , Lesiones del Sistema Vascular/metabolismo , Factores de Edad , Envejecimiento/inmunología , Envejecimiento/patología , Animales , Apoptosis , Adhesión Celular , Células Cultivadas , Quimiotaxis , Ácido Clodrónico/farmacología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hiperplasia , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Masculino , Monocitos/inmunología , Monocitos/metabolismo , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/patología , Comunicación Paracrina/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Transducción de Señal , Factores de Tiempo , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/inmunología , Lesiones del Sistema Vascular/patología , Lesiones del Sistema Vascular/prevención & controlRESUMEN
Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn(2+) additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1(-/-) neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn(2+)-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn(2+) induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.
Asunto(s)
Benzoatos/farmacología , Membrana Celular/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno de Macrófago-1/metabolismo , Tiohidantoínas/farmacología , Citoesqueleto de Actina/metabolismo , Secuencia de Aminoácidos , Animales , Benzoatos/química , Adhesión Celular , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/química , Leucocitos/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno de Macrófago-1/química , Manganeso/metabolismo , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Neutrófilos/metabolismo , Unión Proteica , Tiohidantoínas/químicaRESUMEN
Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high-throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique.
Asunto(s)
Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas , Animales , Ligandos , Ratones , Ratones Endogámicos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunologíaRESUMEN
We report the identification of novel small molecule agonists of integrin CD11b/CD18, which increased, in a dose-dependent manner, the adhesion of the integrin CD11b/CD18 expressing cells to two physiologically relevant ligands: Fibrinogen and iC3b. Compound 6 showed an ex vivo EC(50) of 10.5 microM and in vitro selectivity for binding to the recombinant alphaA-domain of CD11b/CD18. In silico docking experiments suggest that the compounds recognized a hydrophobic cleft in the ligand-binding alphaA-domain, implying an allosteric mechanism of modulation of integrin affinity by this novel compound.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Antígeno CD11b/efectos de los fármacos , Antígenos CD18/efectos de los fármacos , Furanos/química , Tiazolidinedionas/química , Antiinflamatorios no Esteroideos/farmacología , Antígeno CD11b/química , Antígeno CD11b/metabolismo , Antígenos CD18/química , Antígenos CD18/metabolismo , Células CACO-2 , Adhesión Celular/efectos de los fármacos , Furanos/farmacología , Humanos , Conformación Proteica , Relación Estructura-Actividad , Tiazolidinedionas/farmacologíaRESUMEN
BACKGROUND: Multi-organ failure occurs during critical illness and is mediated in part by destructive neutrophil-to-endothelial interactions. The ß2 integrin receptor, CR3 (complement receptor 3; Mac-1; CD11b/CD18), which binds endothelial intercellular adhesion molecule-1 (ICAM-1), plays a key role in promoting the adhesion of activated neutrophils to inflamed endothelia which, when prolonged and excessive, can cause vascular damage. Leukadherin-1 (LA-1) is a small molecule allosteric activator of CR3 and has been shown to promote adhesion of blood neutrophils to inflamed endothelium and restrict tissue infiltration. Therefore, LA-1 offers a novel mechanism of anti-inflammatory action by activation, rather than inhibition, of the neutrophil CR3 integrin. However, whether promotion of neutrophil-to-endothelial interaction by this novel therapeutic is of benefit or detriment to endothelial barrier function is not known. METHODS: Critically ill septic and trauma patients were prospectively enrolled from the surgical and the trauma ICU. Blood was collected from these patients and healthy volunteers. Neutrophils were isolated by dextran sedimentation and adhered to TNF-α (tumor necrosis factor-α)-activated human umbilical vein endothelial (HUVEC) monolayers in the presence or absence of fMLP (formylmethionine-leucine-phenylalanine) and/or LA-1. Electric cell-substrate impedance sensing (ECIS) and exposure of underlying collagen were used to quantify endothelial barrier function and permeability. RESULTS: Neutrophils from critically ill trauma and septic patients caused similar degrees of endothelial barrier disruption which exceeded that caused by cells obtained from healthy controls both kinetically and quantitatively. LA-1 protected barrier function in the absence and presence of fMLP which served as a secondary stimulant to cause maximal loss of barrier function. LA-1 protection was also observed by quantifying collagen exposure underlying endothelial cells challenged with fMLP-stimulated neutrophils. LA-1 treatment resulted in decreased migration dynamics of neutrophils crawling on an endothelial monolayer with reduced speed (µm/s = 0.25 ± 0.01 vs. 0.06 ± 0.01, p < 0.05), path length (µm = 199.5 ± 14.3 vs. 42.1 ± 13.0, p < 0.05), and displacement (µm = 65.2 ± 4.7 vs. 10.4 ± 1.3; p < 0.05). CONCLUSION: Neutrophils from patients with trauma or sepsis cause endothelial barrier disruption to a similar extent relative to each other. The CR3 agonist LA-1 protects endothelial barrier function from damage caused by neutrophils obtained from both populations of critically ill patients even when exposed to secondary stimulation.
RESUMEN
Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-ß, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.
Asunto(s)
Antígeno CD11b/inmunología , Lupus Eritematoso Sistémico/inmunología , Macrófagos/inmunología , Receptores Toll-Like/inmunología , Animales , Antígeno CD11b/genética , Femenino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/inmunología , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos MRL lpr , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Receptores Toll-Like/genéticaRESUMEN
Kidney allograft rejection is associated with infiltration of inflammatory CD11b+ leukocytes. A CD11b agonist leukadherin-1 (LA1) increases leukocyte adhesion, preventing their transmigration and tissue recruitment in vivo. Here, we test the extent to which LA1-mediated activation of CD11b/CD18 enhances kidney allograft survival in a mouse model of fully MHC-mismatched orthotopic kidney transplantation, where C57BL/6J (H-2(b)) recipients received kidney allografts from Balb/c mice (H-2(d)). Isograft control recipients received a kidney from a littermate. Control isograft and allograft recipients were treated daily with cyclosporine (CsA) for 2 weeks, while the test group received CsA therapy and daily LA1 injections during week 1 and alternate days during weeks 2-8. LA1 treatment reduced interstitial leukocyte infiltration in the allograft, reduced neointimal hyperplasia and glomerular damage, and prolonged graft survival from 48.5% (CsA only) to 100% (CsA and LA1) on day 60. Serum creatinine levels showed significantly improved kidney function in LA1-treated mice compared to CsA-treated allograft controls [0.52 ± 0.18 mg/dL vs 0.24 ± 0.07 mg/dL (n = 5), respectively]. Furthermore, combination therapy reduced macrophage infiltration and increased the frequency of FoxP3 + Tregs in the allograft. These findings indicate a crucial role for CD11b/CD18 in the control of leukocyte migration to the transplanted kidney and identify integrin agonist LA1 as a novel potential therapeutic agent for kidney transplantation.
RESUMEN
The BclXL apoptotic repressor bears the propensity to associate into megadalton oligomers in solution, particularly under acidic pH. Herein, using various biophysical methods, we analyze the effect of temperature on the oligomerization of BclXL. Our data show that BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils with length in the order of mm and a diameter in the µm-range under elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely α-helical fold into a predominantly ß-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL fibrils formed under elevated temperatures show no observable affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL fibrils likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, our study sheds light on the propensity of BclXL to form amyloid-like fibrils with important consequences on its mechanism of action in gauging the apoptotic fate of cells in health and disease.
Asunto(s)
Calor , Proteína bcl-X/química , Apoptosis , Cardiolipinas/metabolismo , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
The integrin CD11b/CD18 (also known as Mac-1), which is a heterodimer of the α(M) (CD11b) and ß(2) (CD18) subunits, is critical for leukocyte adhesion and migration and for immune functions. Blocking integrin-mediated leukocyte adhesion, although beneficial in experimental models, has had limited success in treating inflammatory diseases in humans. Here, we used an alternative strategy of inhibiting leukocyte recruitment by activating CD11b/CD18 with small-molecule agonists, which we term leukadherins. These compounds increased the extent of CD11b/CD18-dependent cell adhesion of transfected cells and of primary human and mouse neutrophils, which resulted in decreased chemotaxis and transendothelial migration. Leukadherins also decreased leukocyte recruitment and reduced arterial narrowing after injury in rats. Moreover, compared to a known integrin antagonist, leukadherins better preserved kidney function in a mouse model of experimental nephritis. Leukadherins inhibited leukocyte recruitment by increasing leukocyte adhesion to the inflamed endothelium, which was reversed with a blocking antibody. Thus, we propose that pharmacological activation of CD11b/CD18 offers an alternative therapeutic approach for inflammatory diseases.
Asunto(s)
Adhesión Celular/fisiología , Inflamación/tratamiento farmacológico , Leucocitos/fisiología , Antígeno de Macrófago-1/metabolismo , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Animales , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Calcio , Adhesión Celular/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/fisiología , Humanos , Células K562 , Leucocitos/citología , Antígeno de Macrófago-1/uso terapéutico , Magnesio , Manganeso , Ratones , RatasRESUMEN
During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin alphaIIbbeta3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin alphaIIbbeta3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin alphaIIbbeta3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future.