RESUMEN
Chlamydia trachomatis-genital infection in women can be modeled in mice using Chlamydia muridarum. Using this model, it has been shown that the cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1α lead to irreversible tissue damage in the oviducts. In this study, we investigated the contribution of TNFα on IL-1α synthesis in infected epithelial cells. We show that C muridarum infection enhanced TNFα-induced IL-1α expression and release in a mouse epithelial cell line. In addition to IL-1α, several TNFα-induced inflammatory genes were also highly induced, and infection enhanced TNF-induced cell death. In the mouse model of genital infection, oviducts from mice lacking the TNFα receptor displayed minimal staining for IL-1α compared with wild-type oviducts. Our results suggest TNFα and IL-1α enhance each other's downstream effects resulting in a hyperinflammatory response to chlamydial infection. We propose that biologics targeting TNF-induced IL-1α synthesis could be used to mitigate tissue damage during chlamydial infection.
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Muerte Celular , Infecciones por Chlamydia , Chlamydia muridarum/inmunología , Interleucina-1alfa , Factor de Necrosis Tumoral alfa , Animales , Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/metabolismo , Células Epiteliales , Femenino , Interleucina-1alfa/inmunología , Interleucina-1alfa/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND AND AIMS: Maternal hypercholesterolemia has been implicated in earlier onset of atherosclerotic lesions in neonatal offspring. In this study, we investigated whether maternal exposure to soy protein isolate (SPI) diet attenuated the progression of atherosclerosis in F1 offspring. METHOD: Pregnant apolipoprotein E knockout (Apoe-/-) female mice were fed SPI diet until postnatal day 21 (PND21) of the offspring (SPI-offspring). SPI-offspring were switched at PND21 to casein (CAS) diet until PND140. Mice fed CAS throughout their lifetime (gestation to adulthood) were used as controls (CAS-offspring). RESULTS: Atherosclerotic lesions in the aortic sinuses were reduced in SPI-offspring compared with CAS-offspring. Total serum cholesterol levels in CAS-offspring or dams were comparable to levels in their SPI-counterparts, suggesting that alternative mechanisms contributed to the athero-protective effect of maternal SPI diet. Aortic VCAM-1, MCP-1, and TNF-α mRNA and protein expression, and expression of macrophage pro-inflammatory cytokines was reduced in SPI-offspring. Interestingly, CD4+ T cells from SPI-offspring showed reduced IFN-γ expression (Th1), while the expression of IL-10 (Th2/Treg), and IL-13 (Th2) was increased. DNA methylation analyses revealed that anti-inflammatory T cell-associated Gata3 and Il13 promoter regions were hypomethylated in SPI-offspring. These findings suggest that anti-inflammatory macrophage and T cell response may have contributed to the athero-protective effect in SPI-offspring. CONCLUSIONS: Our findings demonstrate that gestational and lactational soy diet exposure inhibits susceptibility to atherosclerotic lesion formation by promoting anti-inflammatory responses by macrophages and T cells.
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Exposición Materna , Placa Aterosclerótica , Adulto , Animales , Antiinflamatorios , Dieta , Femenino , Humanos , Macrófagos , Ratones , EmbarazoRESUMEN
BACKGROUND AND AIMS: Atherosclerosis is a chronic inflammatory disease, and recent studies have shown that infection at remote sites can contribute to the progression of atherosclerosis in hyperlipidemic mouse models. In this report, we tested the hypothesis that genital Chlamydia infection could accelerate the onset and progression of atherosclerosis. METHODS: Apolipoprotein E (Apoe-/-) and LDL receptor knockout (Ldlr-/-) mice on a high-fat diet were infected intra-vaginally with Chlamydia muridarum. Atherosclerotic lesions on the aortic sinuses and in the descending aorta were assessed at 8-weeks post-infection. Systemic, macrophage, and vascular site inflammatory responses were assessed and quantified. RESULTS: Compared to the uninfected groups, infected Apoe-/- and Ldlr-/- mice developed significantly more atherosclerotic lesions in the aortic sinus and in the descending aorta. Increased lesions were associated with higher circulating levels of serum amyloid A-1, IL-1ß, TNF-α, and increased VCAM-1 expression in the aortic sinus, suggesting an association with inflammatory responses observed during C. muridarum infection. Genital infection courses were similar in Apoe-/-, Ldlr-/-, and wild type mice. Further, Apoe-/- mice developed severe uterine pathology with increased dilatations. Apoe-deficiency also augmented cytokine/chemokine response in C. muridarum infected macrophages, suggesting that the difference in macrophage response could have contributed to the genital pathology in Apoe-/- mice. CONCLUSIONS: Overall, these studies demonstrate that genital Chlamydia infection exacerbates atherosclerotic lesions in hyperlipidemic mouse and suggest a novel role for Apoe in full recovery of uterine anatomy after chlamydial infection.
Asunto(s)
Enfermedades de la Aorta/etiología , Aterosclerosis/etiología , Infecciones por Chlamydia/complicaciones , Chlamydia muridarum/patogenicidad , Hiperlipidemias/complicaciones , Infecciones del Sistema Genital/complicaciones , Útero/microbiología , Animales , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/microbiología , Enfermedades de la Aorta/patología , Aterosclerosis/metabolismo , Aterosclerosis/microbiología , Aterosclerosis/patología , Células Cultivadas , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/patología , Citocinas/sangre , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Hiperlipidemias/metabolismo , Mediadores de Inflamación/sangre , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones Noqueados para ApoE , Placa Aterosclerótica , Receptores de LDL/deficiencia , Receptores de LDL/genética , Infecciones del Sistema Genital/microbiología , Infecciones del Sistema Genital/patología , Factores de Tiempo , Útero/patologíaRESUMEN
Exposure of mice to a diet containing 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) induces porphyrin accumulation, cholestasis, immune response, and hepatobiliary damage mimicking hepatic porphyria and sclerosing cholangitis. Although ß-catenin signaling promotes hepatocyte proliferation, and macrophages are a source of Wnts, the role of macrophage-derived Wnts in modulating hepatobiliary injury/repair remains unresolved. We investigated the effect of macrophage-specific deletion of Wntless, a cargo protein critical for cellular Wnt secretion, by feeding macrophage-Wntless-knockout (Mac-KO) and wild-type littermates a DDC diet for 14 days. DDC exposure induced Wnt11 up-regulation in macrophages. Mac-KO mice on DDC showed increased serum alkaline phosphatase, aspartate aminotransferase, direct bilirubin, and histologic evidence of more cell death, inflammation, and ductular reaction. There was impaired hepatocyte proliferation evidenced by Ki-67 immunostaining, which was associated with decreased hepatocyte ß-catenin activation and cyclin-D1 in Mac-KO. Mac-KO also showed increased CD45, F4/80, and neutrophil infiltration after DDC diet, along with increased expression of several proinflammatory cytokines and chemokines. Gene expression analyses of bone marrow-derived macrophages from Mac-KO mice and F4/80+ macrophages isolated from DDC-fed Mac-KO livers showed proinflammatory M1 polarization. In conclusion, this study shows that a lack of macrophage Wnt secretion leads to more DDC-induced hepatic injury due to impaired hepatocyte proliferation and increased M1 macrophages, which promotes immune-mediated cell injury.
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Colangitis Esclerosante/metabolismo , Colestasis/metabolismo , Dieta/efectos adversos , Hepatocitos/metabolismo , Macrófagos/metabolismo , Piridinas/toxicidad , Proteínas Wnt/biosíntesis , Animales , Colangitis Esclerosante/inducido químicamente , Colangitis Esclerosante/genética , Colangitis Esclerosante/patología , Colestasis/inducido químicamente , Colestasis/genética , Colestasis/patología , Hepatocitos/patología , Macrófagos/patología , Ratones , Ratones Noqueados , Regulación hacia Arriba/efectos de los fármacos , Proteínas Wnt/genéticaRESUMEN
Platelet-derived growth factor receptor α (PDGFRα), a tyrosine kinase receptor, is up-regulated in hepatic stellate cells (HSCs) during chronic liver injury. HSCs mediate hepatic fibrosis through their activation from a quiescent state partially in response to profibrotic growth factors. HSC activation entails enhanced expression of profibrotic genes, increase in proliferation, and increase in motility, which facilitates migration within the hepatic lobule. We show colocalization of PDGFRα in murine carbon tetrachloride, bile duct ligation, and 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine models of chronic liver injury, and investigate the role of PDGFRα on proliferation, profibrotic gene expression, and migration in primary human HSCs (HHSteCs) using the PDGFRα-specific inhibitory monoclonal antibody olaratumab. Although lacking any effects on HHSteC transdifferentiation assessed by gene expression of ACTA2, TGFB1, COL1A1, SYP1, and FN1, olaratumab specifically reduced HHSteC proliferation (AlamarBlue assay) and cell migration (transwell migration assays). Using phospho-specific antibodies, we show that olaratumab attenuates PDGFRα activation in response to PDGF-BB, and reduced phosphorylation of extracellular signal-regulated kinase 1 and 2, Elk-1, p38, Akt, focal adhesion kinase, mechanistic target of rapamycin, C10 regulator of kinase II, and C10 regulator of kinase-like, suggesting that PDGFRα contributes to mitogenesis and actin reorganization through diverse downstream effectors. Our findings support a distinct contribution of PDGFRα signaling to HSC proliferation and migration and provide evidence that inhibition of PDGFRα signaling could alter the pathogenesis of hepatic fibrosis.
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Movimiento Celular , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Comunicación Autocrina/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Densitometría , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Ligandos , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones Endogámicos C57BL , Modelos Biológicos , Fosforilación/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
An important role for ß-catenin in regulating p65 (a subunit of NF-κB) during acute liver injury has recently been elucidated through use of conditional ß-catenin knockout mice, which show protection from apoptosis through increased activation of p65. Thus, we hypothesized that the p65/ß-catenin complex may play a role in regulating processes such as cell proliferation during liver regeneration. We show through in vitro and in vivo studies that the p65/ß-catenin complex is regulated through the TNF-α pathway and not through Wnt signaling. However, this complex is unchanged after partial hepatectomy (PH), despite increased p65 and ß-catenin nuclear translocation as well as cyclin D1 activation. We demonstrate through both in vitro silencing experiments and chromatin immunoprecipitation after PH that ß-catenin, and not p65, regulates cyclin D1 expression. Conversely, using reporter mice we show p65 is activated exclusively in the nonparenchymal (NPC) compartment during liver regeneration. Furthermore, stimulation of macrophages by TNF-α induces activation of NF-κB and subsequent secretion of Wnts essential for ß-catenin activation in hepatocytes. Thus, we show that ß-catenin and p65 are activated in separate cellular compartments during liver regeneration, with p65 activity in NPCs contributing to the activation of hepatocyte ß-catenin, cyclin D1 expression, and subsequent proliferation.
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Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Regulación de la Expresión Génica , Regeneración Hepática , Hígado/metabolismo , Factor de Transcripción ReIA/metabolismo , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular Tumoral , Proliferación Celular , Ciclina D1/metabolismo , Hepatectomía , Hepatocitos/metabolismo , Humanos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Células RAW 264.7 , Factor de Transcripción ReIA/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
BACKGROUND & AIMS: Iron overload disorders such as hereditary hemochromatosis and iron loading anemias are a common cause of morbidity from liver diseases and increase risk of hepatic fibrosis and hepatocellular carcinoma (HCC). Treatment options for iron-induced damage are limited, partly because there is lack of animal models of human disease. Therefore, we investigated the effect of iron overload in liver-specific ß-catenin knockout mice (KO), which are susceptible to injury, fibrosis and tumorigenesis following chemical carcinogen exposure. METHODS: Iron overload diet was administered to KO and littermate control (CON) mice for various times. To ameliorate an oxidant-mediated component of tissue injury, N-Acetyl-L-(+)-cysteine (NAC) was added to drinking water of mice on iron overload diet. RESULTS: KO on iron diet (KO +Fe) exhibited remarkable inflammation, followed by steatosis, oxidative stress, fibrosis, regenerating nodules and occurrence of occasional HCC. Increased injury in KO +Fe was associated with activated protein kinase B (AKT), ERK, and NF-κB, along with reappearance of ß-catenin and target gene Cyp2e1, which promoted lipid peroxidation and hepatic damage. Addition of NAC to drinking water protected KO +Fe from hepatic steatosis, injury and fibrosis, and prevented activation of AKT, ERK, NF-κB and reappearance of ß-catenin. CONCLUSIONS: The absence of hepatic ß-catenin predisposes mice to hepatic injury and fibrosis following iron overload, which was reminiscent of hemochromatosis and associated with enhanced steatohepatitis and fibrosis. Disease progression was notably alleviated by antioxidant therapy, which supports its chemopreventive role in the management of chronic iron overload disorders. LAY SUMMARY: Lack of animal models for iron overload disorders makes it hard to study the disease process for improving therapies. Feeding high iron diet to mice that lack the ß-catenin gene in liver cells led to increased inflammation followed by fat accumulation, cell death and wound healing that mimicked human disease. Administration of an antioxidant prevented hepatic injury in this model.
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Hígado Graso/etiología , Hígado Graso/metabolismo , Sobrecarga de Hierro/complicaciones , Sobrecarga de Hierro/metabolismo , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , beta Catenina/deficiencia , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Modelos Animales de Enfermedad , Hígado Graso/prevención & control , Femenino , Hemocromatosis/complicaciones , Hemocromatosis/metabolismo , Humanos , Sobrecarga de Hierro/tratamiento farmacológico , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/prevención & control , Masculino , Ratones , Ratones Noqueados , Estrés Oxidativo , Transducción de Señal , beta Catenina/genéticaRESUMEN
AIMS: Macropinocytosis has been implicated in cardiovascular and other disorders, yet physiological factors that initiate fluid-phase internalization and the signaling mechanisms involved remain poorly identified. The present study was designed to examine whether matrix protein thrombospondin-1 (TSP1) stimulates macrophage macropinocytosis and, if so, to investigate the potential signaling mechanism involved. RESULTS: TSP1 treatment of human and murine macrophages stimulated membrane ruffle formation and pericellular solute internalization by macropinocytosis. Blockade of TSP1 cognate receptor CD47 and NADPH oxidase 1 (Nox1) signaling, inhibition of phosphoinositide 3-kinase, and transcriptional knockdown of myotubularin-related protein 6 abolished TSP1-induced macropinocytosis. Our results demonstrate that Nox1 signaling leads to dephosphorylation of actin-binding protein cofilin at Ser-3, actin remodeling, and macropinocytotic uptake of unmodified native low-density lipoprotein (nLDL), leading to foam cell formation. Finally, peritoneal chimera studies suggest the role of CD47 in macrophage lipid macropinocytosis in hypercholesterolemic ApoE-/- mice in vivo. INNOVATION: Activation of a previously unidentified TSP1-CD47 signaling pathway in macrophages stimulates direct receptor-independent internalization of nLDL, leading to significant lipid accumulation and foam cell formation. These findings reveal a new paradigm in which delimited Nox1-mediated redox signaling, independent of classical lipid oxidation, contributes to early propagation of vascular inflammatory disease. CONCLUSIONS: The findings of the present study demonstrate a new mechanism of solute uptake with implications for a wide array of cell types, including macrophages, dendritic cells, and cancer cells, and multiple pathological conditions in which matrix proteins are upregulated. Antioxid. Redox Signal. 26, 886-901.
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Factores Despolimerizantes de la Actina/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Antígeno CD47/metabolismo , Hipercolesterolemia/metabolismo , Macrófagos/citología , NADPH Oxidasa 1/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Macrófagos/metabolismo , Ratones , Fosforilación , Pinocitosis , Mapas de Interacción de Proteínas , Células RAW 264.7 , Transducción de Señal , Células THP-1RESUMEN
Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate-tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up-regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low-density lipoprotein-related protein 5/6, transgenic mice expressing stable ß-catenin, and in vitro studies, we show a role of Wnt signaling through ß-catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a ß-catenin-independent manner. CONCLUSION: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both ß-catenin-dependent and -independent manners. (Hepatology 2016;64:1652-1666).
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Colestasis Intrahepática , Regeneración Hepática/fisiología , Proteínas Wnt/fisiología , Animales , Línea Celular Tumoral , Transdiferenciación Celular , Hepatocitos , Humanos , Ratones , Transducción de Señal , beta Catenina/fisiologíaRESUMEN
OBJECTIVE: Fcγ receptors (FcγRs) are classified as activating (FcγRI, III, and IV) and inhibitory (FcγRII) receptors. We have reported that deletion of activating FcγRs in apolipoprotein E (apoE) single knockout mice attenuated atherosclerosis. In this report, we investigated the hypothesis that deficiency of inhibitory FcγRIIb exacerbates atherosclerosis. APPROACH AND RESULTS: ApoE-FcγRIIb double knockout mice, congenic to the C57BL/6 (apoE-FcγRIIbB6 (-/-)), were generated and atherosclerotic lesions were assessed. In contrary to our hypothesis, when compared with apoE single knockout mice, arterial lesions were significantly decreased in apoE-FcγRIIbB6 (-/-) male and female mice fed chow or high-fat diets. Chimeric mice generated by transplanting apoE-FcγRIIbB6 (-/-) marrow into apoE single knockout mice also developed reduced lesions. CD4(+) T cells from apoE-FcγRIIbB6 (-/-) mice produced higher levels of interleukin-10 and transforming growth factor-ß than their apoE single knockout counterparts. As our findings conflict with a previous report using apoE-FcγRIIb129/B6 (-/-) mice on a mixed genetic background, we investigated whether strain differences contributed to the anti-inflammatory response. Macrophages from FcγRIIb129/B6 (-/-) mice on a mixed genetic background produced more interleukin-1ß and MCP-1 (monocyte chemoattractant protein-1) in response to immune complexes, whereas congenic FcγRIIbB6 (-/-) mice generated more interleukin-10 and significantly less interleukin-1ß. Interestingly, the expression of lupus-associated slam genes, located in proximity to fcgr2b in mouse chromosome 1, is upregulated only in mixed FcγRIIb129/B6 (-/-) mice. CONCLUSIONS: Our findings demonstrate a detrimental role for FcγRIIb signaling in atherosclerosis and the contribution of anti-inflammatory cytokine responses in the attenuated lesions observed in apoE-FcγRIIbB6 (-/-) mice. As 129/sv genome-derived lupus-associated genes have been implicated in lupus phenotype in FcγRIIb129/B6 (-/-) mice, our findings suggest possible epistatic mechanism contributing to the decreased lesions.
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Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Receptores de IgG/metabolismo , Animales , Aterosclerosis/inmunología , Aterosclerosis/fisiopatología , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distribución Aleatoria , Receptores de IgG/inmunología , Sensibilidad y Especificidad , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Stroke, caused by carotid plaque rupture, is a major cause of death in the United States. Whereas vulnerable human plaques have higher Fc receptor (FcγR) expression than their stable counterparts, how FcγR expression impacts plaque histology is unknown. We investigated the role of FcγRIIb in carotid plaque development and stability in apolipoprotein (Apo)e−/− and Apoe−/−FcγRIIb−/− double knockout (DKO) animals. METHODS AND RESULTS: Plaques were induced by implantation of a shear stressmodifying cast around the carotid artery. Plaque length and stenosis were followed longitudinally using ultrasound biomicroscopy. Immune status was determined by flow cytometry, cytokine release, immunoglobulin G concentration and analysis of macrophage polarization both in plaques and in vitro. Surprisingly, DKO animals had lower plaque burden in both carotid artery and descending aorta. Plaques from Apoe−/− mice were foamcell rich and resembled vulnerable human specimens, whereas those from DKO mice were fibrous and histologically stable. Plaques from DKO animals expressed higher arginase 1 (Arg1) and lower inducible nitric oxide synthase (iNOS), indicating the presence of M2 macrophages. Analysis of blood and cervical lymph nodes revealed higher interleukin (IL)10, immune complexes, and regulatory T cells (Tregs) and lower IL12, IL1ß, and tumor necrosis factor alpha (TNFα) in DKO mice. Similarly, in vitro stimulation produced higher IL10 and Arg1 and lower iNOS, IL1ß, and TNFα in DKO versus Apoe−/− macrophages. These results define a systemic antiinflammatory phenotype. CONCLUSIONS: We hypothesized that removal of FcγRIIb would exacerbate atherosclerosis and generate unstable plaques. However, we found that deletion of FcγRIIb on a congenic C57BL/6 background induces an antiinflammatory Treg/M2 polarization that is atheroprotective.
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Apolipoproteínas E/deficiencia , Arterias Carótidas/metabolismo , Estenosis Carotídea/prevención & control , Inflamación/prevención & control , Placa Aterosclerótica , Receptores de IgG/deficiencia , Animales , Apolipoproteínas E/genética , Arginasa/metabolismo , Arterias Carótidas/diagnóstico por imagen , Arterias Carótidas/inmunología , Estenosis Carotídea/diagnóstico por imagen , Estenosis Carotídea/genética , Estenosis Carotídea/inmunología , Estenosis Carotídea/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Genotipo , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Noqueados , Microscopía Acústica , Necrosis , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fenotipo , Receptores de IgG/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
INTRODUCTION: Sepsis and other infections are associated with late cardiovascular events. Although persistent inflammation is implicated, a causal relationship has not been established. We tested whether sepsis causes vascular inflammation and accelerates atherosclerosis. METHODS: We performed prospective, randomized animal studies at a university research laboratory involving adult male ApoE-deficient (ApoE-/-) and young C57B/L6 wild-type (WT) mice. In the primary study conducted to determine whether sepsis accelerates atherosclerosis, we fed ApoE-/- mice (N = 46) an atherogenic diet for 4 months and then performed cecal ligation and puncture (CLP), followed by antibiotic therapy and fluid resuscitation or a sham operation. We followed mice for up to an additional 5 months and assessed atheroma in the descending aorta and root of the aorta. We also exposed 32 young WT mice to CLP or sham operation and followed them for 5 days to determine the effects of sepsis on vascular inflammation. RESULTS: ApoE-/- mice that underwent CLP had reduced activity during the first 14 days (38% reduction compared to sham; P < 0.001) and sustained weight loss compared to the sham-operated mice (-6% versus +9% change in weight after CLP or sham surgery to 5 months; P < 0.001). Despite their weight loss, CLP mice had increased atheroma (46% by 3 months and 41% increase in aortic surface area by 5 months; P = 0.03 and P = 0.004, respectively) with increased macrophage infiltration into atheroma as assessed by immunofluorescence microscopy (0.52 relative fluorescence units (rfu) versus 0.97 rfu; P = 0.04). At 5 months, peritoneal cultures were negative; however, CLP mice had elevated serum levels of interleukin 6 (IL-6) and IL-10 (each at P < 0.05). WT mice that underwent CLP had increased expression of intercellular adhesion molecule 1 in the aortic lumen versus sham at 24 hours (P = 0.01) that persisted at 120 hours (P = 0.006). Inflammatory and adhesion genes (tumor necrosis factor α, chemokine (C-C motif) ligand 2 and vascular cell adhesion molecule 1) and the adhesion assay, a functional measure of endothelial activation, were elevated at 72 hours and 120 hours in mice that underwent CLP versus sham-operations (all at P <0.05). CONCLUSIONS: Using a combination of existing murine models for atherosclerosis and sepsis, we found that CLP, a model of intra-abdominal sepsis, accelerates atheroma development. Accelerated atheroma burden was associated with prolonged systemic, endothelial and intimal inflammation and was not explained by ongoing infection. These findings support observations in humans and demonstrate the feasibility of a long-term follow-up murine model of sepsis.
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Aterosclerosis/etiología , Sepsis/complicaciones , Abdomen , Animales , Aorta/patología , Aterosclerosis/sangre , Aterosclerosis/patología , Biomarcadores/sangre , Moléculas de Adhesión Celular/metabolismo , Citocinas/sangre , Citocinas/genética , Modelos Animales de Enfermedad , Expresión Génica , Masculino , Ratones Endogámicos C57BL , Estudios Prospectivos , ARN Mensajero/metabolismo , Distribución Aleatoria , Sepsis/sangre , Pérdida de PesoRESUMEN
Recent studies showed loss of CD36 or scavenger receptor-AI/II (SR-A) does not ameliorate atherosclerosis in a hyperlipidemic mouse model, suggesting receptors other than CD36 and SR-A may also contribute to atherosclerosis. In this report, we show that apolipoprotein E (apoE)-CD16 double knockout (DKO; apoE-CD16 DKO) mice have reduced atherosclerotic lesions compared with apoE knockout mice. In vivo and in vitro foam cell analyses showed apoE-CD16 DKO macrophages accumulated less neutral lipids. Reduced foam cell formation in apoE-CD16 DKO mice is not due to change in expression of CD36, SR-A, and LOX-1. This led to a hypothesis that CD16 may have scavenger receptor activity. We presented evidence that a soluble form of recombinant mouse CD16 (sCD16) bound to malondialdehyde-modified low-density lipoprotein (MDALDL), and this binding is blocked by molar excess of MDA- modified BSA and anti-MDA mAbs, suggesting CD16 specifically recognizes MDA epitopes. Interestingly, sCD16 inhibited MDALDL binding to macrophage cell line, as well as soluble forms of recombinant mouse CD36, SR-A, and LOX-1, indicating CD16 can cross-block MDALDL binding to other scavenger receptors. Anti-CD16 mAb inhibited immune complex binding to sCD16, whereas it partially inhibited MDALDL binding to sCD16, suggesting MDALDL binding site may be in close proximity to the immune complex binding site in CD16. Loss of CD16 expression resulted in reduced levels of MDALDL-induced proinflammatory cytokine expression. Finally, CD16-deficient macrophages showed reduced MDALDL-induced Syk phosphorylation. Collectively, our findings suggest scavenger receptor activity of CD16 may, in part, contribute to the progression of atherosclerosis.
Asunto(s)
Apolipoproteínas E/inmunología , Aterosclerosis/inmunología , Hiperlipidemias/inmunología , Receptores de IgG/inmunología , Receptores Depuradores/inmunología , Animales , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Antígenos CD36/genética , Antígenos CD36/inmunología , Hiperlipidemias/genética , Hiperlipidemias/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Ratones , Ratones Noqueados , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/inmunología , Receptores de IgG/genética , Receptores Depuradores/genética , Quinasa SykRESUMEN
PURPOSE: Inflammation is a hallmark of many diseases, such as atherosclerosis, autoimmune diseases, obesity, and cancer. Isoflavone-free soy protein diet (SPI(-)) has been shown to reduce atherosclerotic lesions in a hyperlipidemic mouse model compared to casein (CAS)-fed mice, despite unchanged serum lipid levels. However, possible mechanisms contributing to the athero-protective effect of soy protein remain unknown. Therefore, we investigated whether and how SPI(-) diet inhibits inflammatory responses associated with atherosclerosis. METHODS: Apolipoprotein E knockout (apoE-/-) mice (5-week) were fed CAS or SPI(-) diet for 1 or 5 week to determine LPS- and hyperlipidemia-induced acute and chronic inflammatory responses, respectively. Expression of NF-κB-dependent inflammation mediators such as VCAM-1, TNF-α, and MCP-1 were determined in aorta and liver. NF-κB, MAP kinase, and AKT activation was determined to address mechanisms contributing to the anti-inflammatory properties of soy protein/peptides. RESULTS: Isoflavone-free soy protein diet significantly reduced LPS-induced VCAM-1 mRNA and protein expression in aorta compared to CAS-fed mice. Reduced VCAM-1 expression in SPI(-)-fed mice also paralleled attenuated monocyte adhesion to vascular endothelium, a critical and primary processes during inflammation. Notably, VCAM-1 mRNA and protein expression in lesion-prone aortic arch was significantly reduced in apoE-/- mice fed SPI(-) for 5 weeks compared with CAS-fed mice. Moreover, dietary SPI(-) potently inhibited LPS-induced NF-κB activation and the subsequent upregulation of pro-inflammatory cytokines, including TNF-α, IL-6, IL-1ß, and MCP-1. Interestingly, SPI(-) inhibited NF-κB-dependent inflammatory responses by targeting I-κB phosphorylation and AKT activation with no effect on MAP kinase pathway. Of the five putative soy peptides, four of the soy peptides inhibited LPS-induced VCAM-1, IL-6, IL-8, and MCP-1 protein expression in human vascular endothelial cells in vitro. CONCLUSIONS: Collectively, our findings suggest that anti-inflammatory properties of component(s) of soy protein/peptides may be a possible mechanism for the prevention of chronic inflammatory diseases such as atherosclerosis.
Asunto(s)
Antiinflamatorios/farmacología , Citocinas/metabolismo , Inflamación/tratamiento farmacológico , Proteínas de Soja/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Apolipoproteínas E/genética , Caseínas/administración & dosificación , Adhesión Celular/efectos de los fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lipopolisacáridos/efectos adversos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Diet is highly linked to breast cancer risk, yet little is known about its influence on mammary epithelial populations with distinct regenerative and hence, tumorigenic potential. To investigate this, we evaluated the relative frequency of lineage-negative CD29(hi)CD24(+), CD29(lo)CD24(+) and CD29(hi)Thy1(+)CD24(+) epithelial subpopulations in pre-neoplastic mammary tissue of adult virgin MMTV-Wnt1-transgenic mice fed either control (Casein) or soy-based diets. We found that mammary epithelial cells exposed to soy diet exhibited a lower percentage of CD29(hi)CD24(+)Lin(-) population, decreased ability to form mammospheres in culture, lower mammary outgrowth potential when transplanted into cleared fat pads, and reduced appearance of tumor-initiating CD29(hi)Thy1(+)CD24(+) cells, than in those of control diet-fed mice. Diet had no comparable influence on the percentage of the CD29(lo)CD24(+)Lin(-) population. Global gene expression profiling of the CD29(hi)CD24(+)subpopulation revealed markedly altered expression of genes important to inflammation, cytokine and chemokine signaling, and proliferation. Soy-fed relative to casein-fed mice showed lower mammary tumor incidence, shorter tumor latency, and reduced systemic levels of estradiol 17-ß, progesterone and interleukin-6. Our results provide evidence for the functional impact of diet on specific epithelial subpopulations that may relate to breast cancer risk and suggest that diet-regulated cues can be further explored for breast cancer risk assessment and prevention.
Asunto(s)
Antígeno CD24/metabolismo , Citocinas/genética , Dieta , Células Epiteliales/citología , Integrina beta1/metabolismo , Neoplasias Mamarias Animales/patología , Animales , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/metabolismo , Regulación hacia Abajo , Células Epiteliales/metabolismo , Femenino , Humanos , Masculino , Glándulas Mamarias Humanas/metabolismo , Neoplasias Mamarias Animales/prevención & control , Ratones , Ratones Transgénicos , Receptores Virales/deficiencia , Receptores Virales/genética , Receptores Virales/metabolismo , Factores de Riesgo , Transcriptoma , Proteína Wnt1/deficiencia , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMEN
Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.
Asunto(s)
Anticolesterolemiantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Hipercolesterolemia/sangre , Monocitos/metabolismo , Simvastatina/farmacología , Tromboplastina/metabolismo , Animales , Coagulación Sanguínea/fisiología , Células Cultivadas , Chlorocebus aethiops , Humanos , Lipoproteínas LDL/metabolismo , Masculino , Ratones , Receptores de LDL/genética , Receptores de LDL/metabolismo , Trombosis , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 6/genética , Receptor Toll-Like 6/metabolismoRESUMEN
Recent studies have shown that some flavonoids are modulators of proinflammatory cytokine production. In this study, velutin, a unique flavone isolated from the pulp of açaí fruit (Euterpe oleracea Mart.), was examined for its effects in reducing lipopolysaccharide-induced proinflammatory cytokine tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in RAW 264.7 peripheral macrophages and mice peritoneal macrophages. Three other structurally similar and well-studied flavones, luteolin, apigenin and chrysoeriol, were included as controls and for comparative purposes. Velutin exhibited the greatest potency among all flavones in reducing TNF-α and IL-6 production. Velutin also showed the strongest inhibitory effect in nuclear factor (NF)-κB activation (as assessed by secreted alkaline phosphatase reporter assay) and exhibited the greatest effects in blocking the degradation of inhibitor of NF-κB as well as in inhibiting mitogen-activated protein kinase p38 and JNK phosphorylation; all of these are important signaling pathways involved in production of TNF-α and IL-6. The present study led to the discovery of a strong anti-inflammatory flavone, velutin. This compound effectively inhibited the expression of proinflammatory cytokines TNF-α and IL-6 in low micromole levels by inhibiting NF-κB activation and p38 and JNK phosphorylation.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Flavonas/farmacología , Interleucina-6/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antiinflamatorios no Esteroideos/química , Apigenina/química , Apigenina/farmacología , Arecaceae/química , Línea Celular Transformada , Células Cultivadas , Flavonas/química , Flavonoides/química , Flavonoides/farmacología , Frutas/química , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/genética , Lipopolisacáridos , Luteolina/química , Luteolina/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/agonistas , FN-kappa B/antagonistas & inhibidores , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Though the presence of antioxidized low-density lipoprotein IgG is well documented in clinical and animal studies, the role for FcγRs to the progression of atherosclerosis has not been studied in detail. In the current study, we investigated the role for activating FcγR in the progression of atherosclerosis using apolipoprotein E (apoE)-Fcγ-chain double-knockout (DKO) mice. Relative to apoE knockout (KO) mice, arterial lesion formation was significantly decreased in apoE-Fcγ-chain DKO mice. Bone marrow chimera studies showed reduced lesions in apoE KO mice receiving the bone marrow of apoE-Fcγ-chain DKO mice. Compared to apoE KO mice, antioxidized low-density lipoprotein IgG1 (Th2) and IgG2a (Th1), IL-10, and IFN-γ secretion by activated T cells was increased in apoE-Fcγ-chain DKO mice. These findings suggest that reduced atherosclerotic lesion in apoE-Fcγ-chain DKO mice is not due to a Th1/Th2 imbalance. Interestingly, the number of Th17 cells and the secretion of IL-17 by activated CD4(+) cells were decreased in apoE-Fcγ-chain DKO mice. Notably, the number of regulatory T cells, expression of mRNA, and secretion of TGF-ß and IL-10 were increased in apoE-Fcγ-chain DKO mice. Furthermore, secretions of IL-6 and STAT-3 phosphorylation essential for Th17 cell genesis were reduced in apoE-Fcγ-chain DKO mice. Importantly, decrease in Th17 cells in apoE-Fcγ-chain DKO mice was due to reduced IL-6 release by APC of apoE-Fcγ-chain DKO mice. Collectively, our data suggest that activating FcγR promotes atherosclerosis by inducing a Th17 response in the hyperlipidemic apoE KO mouse model.
Asunto(s)
Aterosclerosis/inmunología , Hiperlipidemias/inmunología , Receptores Fc/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/inmunología , Aterosclerosis/metabolismo , Diferenciación Celular/inmunología , Separación Celular , Modelos Animales de Enfermedad , Citometría de Flujo , Hiperlipidemias/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Fc/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Th17/citologíaRESUMEN
Blueberries (BB) have been reported to attenuate atherosclerosis in apoE-deficient (ApoE(-/-) ) mice. The aim of this study was to evaluate the effects of BB in reducing pro-inflammatory cytokine production in mouse macrophages. ApoE(-/-) mice were fed AIN-93G diet (CD) or CD formulated to contain 1% freeze-dried BB for 5 wk. TNF-α and IL-6 were lower in serum of BB-fed mice and TNF-α expression in aorta was down-regulated with BB feeding. Protein level and mRNA expression of TNF-α and IL-6 were significantly lower in the peritoneal macrophages from mice fed BB without or with LPS or oxLDL stimulation. RAW264.7 macrophages were treated with polyphenol-enriched extracts made from the sera of rats fed CD (SEC) or CD containing 10% BB (SEB). SEB significantly inhibited LPS-induced mRNA expression and protein levels of TNF-α and IL-6. Furthermore, SEB inhibited the phosphorylation of IκB, NF-κB p65, MAPK p38 and JNK. All of these are important signaling pathways involved in the production of TNF-α and IL-6.
Asunto(s)
Arándanos Azules (Planta) , Inflamación/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Femenino , Regulación de la Expresión Génica , Interleucina-6/genética , Ratones , Ratones Mutantes , Ratas , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Blueberries have recently been reported to reduce atherosclerotic lesion progression in apoE deficient (apoE(-/-)) mice. However, the underlying mechanisms are not fully understood. The objective of this study was to determine whether lowbush blueberries altered scavenger receptor expression and foam cell formation in apoE(-/-) mice. ApoE(-/-) mice were fed AIN-93 diet (CD) or CD formulated to contain 1% freeze-dried lowbush blueberries (BB) for 20 weeks. Gene expression and protein levels of scavenger receptor CD36 and SR-A in aorta and thioglycollate-elicited peritoneal macrophages (PM) were lower in mice fed BB (P < 0.05). In the second experiment, apoE(-/-) mice were fed CD or BB for 5 weeks. PM were collected and cultured. Gene expression and protein levels of CD36 and SR-A were found to be lower in PM of BB fed mice (P < 0.05). In PM from BB fed mice, fewer oxLDL-induced foam cells were formed compared to those from mice fed CD. Gene expression and protein levels of PPARγ were lower in the PM of BB fed mice (P < 0.05). Detectable isomers of hydroxyoctadecadienoic acids (HODEs) and hydroxyeicosatetraenoic acid (HETEs) were also lower in the PM of BB fed mice (P < 0.05 or P < 0.01). In conclusion, BB inhibited expression of the two major scavenger receptors CD36 and SR-A in PM of apoE(-/-) mice, at least in part through down-regulating PPARγ and reducing its endogenous ligands HODEs and HETEs. We proposed that BB mediated reduction of scavenger receptor expression and attenuation of oxLDL-induced foam cell formation in PM of apoE(-/-) mice are important mechanisms of the athero-protective effects of BB.