RESUMEN
Uncontrolled inflammatory processes are at the root of numerous pathologies. Most recently, studies on confirmed COVID-19 cases have suggested that mortality might be due to virally induced hyperinflammation. Uncontrolled pro-inflammatory states are often driven by continuous positive feedback loops between pro-inflammatory signaling and oxidative stress, which cannot be resolved in a targeted manner. Here, we report on the development of multidrug nanoparticles for the mitigation of uncontrolled inflammation. The nanoparticles are made by conjugating squalene, a natural lipid, to adenosine, an endogenous immunomodulator, and then encapsulating α-tocopherol, as antioxidant. This resulted in high drug loading, biocompatible, multidrug nanoparticles. By exploiting the endothelial dysfunction at sites of acute inflammation, these multidrug nanoparticles delivered the therapeutic agents in a targeted manner, conferring survival advantage to treated animals in models of endotoxemia. Selectively delivering adenosine and antioxidants together could serve as a novel therapeutic approach for safe treatment of acute paradoxal inflammation.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Endotoxemia/tratamiento farmacológico , Nanopartículas/química , Escualeno/química , Síndrome de Respuesta Inflamatoria Sistémica/tratamiento farmacológico , Adenosina/administración & dosificación , Adenosina/química , Animales , Antioxidantes/administración & dosificación , Antioxidantes/química , Betacoronavirus , COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Endotoxemia/inducido químicamente , Femenino , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/química , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Pandemias , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/patología , Neumonía Viral/virología , SARS-CoV-2 , Escualeno/administración & dosificación , Síndrome de Respuesta Inflamatoria Sistémica/inducido químicamente , Resultado del Tratamiento , alfa-Tocoferol/administración & dosificación , alfa-Tocoferol/químicaRESUMEN
BACKGROUND: The cyclic AMP (adenosine monophosphate; cAMP)-hydrolyzing protein PDE4B (phosphodiesterase 4B) is a key negative regulator of cardiac ß-adrenergic receptor stimulation. PDE4B deficiency leads to abnormal Ca2+ handling and PDE4B is decreased in pressure overload hypertrophy, suggesting that increasing PDE4B in the heart is beneficial in heart failure. METHODS: We measured PDE4B expression in human cardiac tissues and developed 2 transgenic mouse lines with cardiomyocyte-specific overexpression of PDE4B and an adeno-associated virus serotype 9 encoding PDE4B. Myocardial structure and function were evaluated by echocardiography, ECG, and in Langendorff-perfused hearts. Also, cAMP and PKA (cAMP dependent protein kinase) activity were monitored by Förster resonance energy transfer, L-type Ca2+ current by whole-cell patch-clamp, and cardiomyocyte shortening and Ca2+ transients with an Ionoptix system. Heart failure was induced by 2 weeks infusion of isoproterenol or transverse aortic constriction. Cardiac remodeling was evaluated by serial echocardiography, morphometric analysis, and histology. RESULTS: PDE4B protein was decreased in human failing hearts. The first PDE4B-transgenic mouse line (TG15) had a ≈15-fold increase in cardiac cAMP-PDE activity and a ≈30% decrease in cAMP content and fractional shortening associated with a mild cardiac hypertrophy that resorbed with age. Basal ex vivo myocardial function was unchanged, but ß-adrenergic receptor stimulation of cardiac inotropy, cAMP, PKA, L-type Ca2+ current, Ca2+ transients, and cell contraction were blunted. Endurance capacity and life expectancy were normal. Moreover, these mice were protected from systolic dysfunction, hypertrophy, lung congestion, and fibrosis induced by chronic isoproterenol treatment. In the second PDE4B-transgenic mouse line (TG50), markedly higher PDE4B overexpression, resulting in a ≈50-fold increase in cardiac cAMP-PDE activity caused a ≈50% decrease in fractional shortening, hypertrophy, dilatation, and premature death. In contrast, mice injected with adeno-associated virus serotype 9 encoding PDE4B (1012 viral particles/mouse) had a ≈50% increase in cardiac cAMP-PDE activity, which did not modify basal cardiac function but efficiently prevented systolic dysfunction, apoptosis, and fibrosis, while attenuating hypertrophy induced by chronic isoproterenol infusion. Similarly, adeno-associated virus serotype 9 encoding PDE4B slowed contractile deterioration, attenuated hypertrophy and lung congestion, and prevented apoptosis and fibrotic remodeling in transverse aortic constriction. CONCLUSIONS: Our results indicate that a moderate increase in PDE4B is cardioprotective and suggest that cardiac gene therapy with PDE4B might constitute a new promising approach to treat heart failure.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Expresión Génica , Insuficiencia Cardíaca/etiología , Miocardio/metabolismo , Remodelación Ventricular/genética , Agonistas Adrenérgicos beta/farmacología , Animales , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Terapia Genética , Vectores Genéticos/genética , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Pruebas de Función Cardíaca , Humanos , Isoproterenol/farmacología , Ratones , Ratones Transgénicos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fenotipo , Receptores Adrenérgicos beta/metabolismo , Transducción Genética , Remodelación Ventricular/efectos de los fármacosRESUMEN
Neutrophils are essential during contact hypersensitivity (CHS), a common skin allergic disease. NF-E2-related factor-2 (Nrf2) is a key regulator of redox balance and skin homeostasis playing a protective role in CHS. In this study, we investigated Nrf2 role in neutrophil recruitment during the sensitization phase of CHS. Comparing wild-type and Nrf2 knockout mice, we demonstrated that Nrf2 regulated dinitrochlorobenzene-induced xenoinflammation, notably neutrophil recruitment to sensitized skin. Nrf2 protective role was associated with high expression of antioxidant genes (ho-1, gclc, nqo1 ) and decreased chemokine production (CCL2, CCL4, CCL11). Interestingly, skin sensitization induced CD36 upregulation in skin-resident macrophages. In vitro results confirmed that the transcription of cd36 gene in macrophages was dependent on Nrf2 and led to an improved capacity to phagocyte-damaged neutrophils by efferocytosis. Nrf2 emerges as a critical target in the sensitization phase of CHS regulating neutrophil recruitment and accumulation in the skin through antioxidant-dependent and -independent mechanisms.
Asunto(s)
Dermatitis por Contacto/inmunología , Regulación de la Expresión Génica/inmunología , Factor 2 Relacionado con NF-E2/inmunología , Infiltración Neutrófila , Neutrófilos/inmunología , Piel/inmunología , Animales , Antioxidantes , Quimiocinas/genética , Quimiocinas/inmunología , Dermatitis por Contacto/genética , Dermatitis por Contacto/patología , Ratones , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Neutrófilos/patología , Piel/patologíaRESUMEN
Rheumatoid arthritis (RA) is a prevalent autoimmune disease characterized by joint inflammation, bone and cartilage erosion. The use of glucocorticoids in the treatment of RA is hampered by significant side effects induced by their unfavorable pharmacokinetics. Delivering glucocorticoids by means of nanotechnologies is promising but the encapsulation of highly crystalline and poorly water-soluble drugs results in poor loading and low stability. We report here the design of 130â¯nm nanoparticles made of solely dexamethasone palmitate, stabilized by polyethylene glycol-linked phospholipids displaying a negative zeta potential (-55â¯mV), high entrapment efficiency and stability over 21â¯days under storage at 4⯰C. X ray diffraction showed no crystallization of the drug. When incubated in serum, nanoparticles released free dexamethasone which explains the in vitro anti-inflammatory effect on LPS-activated RAW 264.7 macrophages. Moreover, we demonstrate in a murine collagen-induced arthritis model the improved therapeutic efficacy of these nanoparticles. Their passive accumulation in arthritic joints leads to disease remission and recovery of the joint structure at a dose of 1â¯mg/kg dexamethasone, without any adverse effects. Dexamethasone palmitate nanoparticles are promising in the treatment of inflammation in rheumatoid arthritis with a very significant difference occurring at the late stage of inflammation allowing to prevent the progression of the disease.
Asunto(s)
Antiinflamatorios/administración & dosificación , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Dexametasona/administración & dosificación , Portadores de Fármacos/administración & dosificación , Nanopartículas/administración & dosificación , Palmitatos/administración & dosificación , Animales , Artritis Experimental/patología , Artritis Reumatoide/patología , Articulaciones/efectos de los fármacos , Articulaciones/patología , Masculino , Ratones , Ratones Endogámicos DBA , Células RAW 264.7RESUMEN
Clostridium difficile infection (CDI) is a major healthcare-associated disease with high recurrence rates. Host colonization is critical for the infectious process, both in first episodes and in recurrent disease, with biofilm formation playing a key role. The ability of C. difficile to form a biofilm on abiotic surfaces is established, but has not yet been confirmed in the intestinal tract. Here, four different isolates of C. difficile, which are in vitro biofilm producers, were studied for their ability to colonize germ-free mice. The level of colonization achieved was similar for all isolates in the different parts of the murine gastrointestinal tract, but pathogen burden was higher in the cecum and colon. Confocal laser scanning microscopy revealed that C. difficile bacteria were distributed heterogeneously over the intestinal tissue, without contact with epithelial cells. The R20291 strain, which belongs to the Ribotype 027 lineage, displayed a unique behavior compared to the other strains by forming numerous aggregates. By immunochemistry analyses, we showed that bacteria were localized inside and outside the mucus layer, irrespective of the strains tested. Most bacteria were entrapped in 3-D structures overlaying the mucus layer. For the R20291 strain, the cell-wall associated polysaccharide PS-II was detected in large amounts in the 3-D structure. As this component has been detected in the extrapolymeric matrix of in vitro C. difficile biofilms, our data suggest strongly that at least the R20291 strain is organized in the mono-associated mouse model in glycan-rich biofilm architecture, which sustainably maintains bacteria outside the mucus layer.
Asunto(s)
Quimiocina CXCL12/fisiología , Interacciones Huésped-Patógeno , Infecciones por Papillomavirus , Receptores CXCR4/fisiología , Epidermodisplasia Verruciforme/genética , Epidermodisplasia Verruciforme/inmunología , Epidermodisplasia Verruciforme/virología , Predisposición Genética a la Enfermedad , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/virología , Mutación , Papillomaviridae/inmunología , Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/inmunología , Factores de Riesgo , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
The CXCL12/CXCR4 signaling exerts a dominant role in promoting hematopoietic stem and progenitor cell (HSPC) retention and quiescence in bone marrow. Gain-of-function CXCR4 mutations that affect homologous desensitization of the receptor have been reported in the WHIM Syndrome (WS), a rare immunodeficiency characterized by lymphopenia. The mechanisms underpinning this remain obscure. Using a mouse model with a naturally occurring WS-linked gain-of-function Cxcr4 mutation, we explored the possibility that the lymphopenia in WS arises from defects at the HSPC level. We reported that Cxcr4 desensitization is required for quiescence/cycling balance of murine short-term hematopoietic stem cells and their differentiation into multipotent and downstream lymphoid-biased progenitors. Alteration in Cxcr4 desensitization resulted in decrease of circulating HSPCs in five patients with WS. This was also evidenced in WS mice and mirrored by accumulation of HSPCs in the spleen, where we observed enhanced extramedullary hematopoiesis. Therefore, efficient Cxcr4 desensitization is critical for lymphoid differentiation of HSPCs, and its impairment is a key mechanism underpinning the lymphopenia observed in mice and likely in WS patients.
Asunto(s)
Diferenciación Celular/genética , Células Madre Hematopoyéticas/metabolismo , Linfocitos/metabolismo , Receptores CXCR4/genética , Adulto , Animales , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea/métodos , Supervivencia Celular/genética , Niño , Citometría de Flujo , Expresión Génica , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/metabolismo , Recuento de Linfocitos , Ratones Transgénicos , Mutación , Enfermedades de Inmunodeficiencia Primaria , Receptores CXCR4/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Bazo/citología , Bazo/metabolismo , Verrugas/genética , Verrugas/metabolismoRESUMEN
BACKGROUND & AIMS: Alcoholic liver disease (ALD) is a leading cause of liver failure and mortality. In humans, severe alcoholic hepatitis is associated with key changes to intestinal microbiota (IM), which influences individual sensitivity to develop advanced ALD. We used the different susceptibility to ALD observed in two distinct animal facilities to test the efficiency of two complementary strategies (fecal microbiota transplantation and prebiotic treatment) to reverse dysbiosis and prevent ALD. METHODS: Mice were fed alcohol in two distinct animal facilities with a Lieber DeCarli diet. Fecal microbiota transplantation was performed with fresh feces from alcohol-resistant donor mice to alcohol-sensitive receiver mice three times a week. Another group of mice received pectin during the entire alcohol consumption period. RESULTS: Ethanol induced steatosis and liver inflammation, which were associated with disruption of gut homeostasis, in alcohol-sensitive, but not alcohol resistant mice. IM analysis showed that the proportion of Bacteroides was specifically lower in alcohol-sensitive mice (p<0.05). Principal coordinate analysis showed that the IM of sensitive and resistant mice clustered differently. We targeted IM using two different strategies to prevent alcohol-induced liver lesions: (1) pectin treatment which induced major modifications of the IM, (2) fecal microbiota transplantation which resulted in an IM very close to that of resistant donor mice in the sensitive recipient mice. Both methods prevented steatosis, liver inflammation, and restored gut homeostasis. CONCLUSIONS: Manipulation of IM can prevent alcohol-induced liver injury. The IM should be considered as a new therapeutic target in ALD. LAY SUMMARY: Sensitivity to alcoholic liver disease (ALD) is driven by intestinal microbiota in alcohol fed mice. Treatment of mice with alcohol-induced liver lesions by fecal transplant from alcohol fed mice resistant to ALD or with prebiotic (pectin) prevents ALD. These findings open new possibilities for treatment of human ALD through intestinal microbiota manipulation.
Asunto(s)
Disbiosis/microbiología , Disbiosis/prevención & control , Microbioma Gastrointestinal/fisiología , Hepatopatías Alcohólicas/microbiología , Hepatopatías Alcohólicas/prevención & control , Animales , Bacteroides/genética , Bacteroides/aislamiento & purificación , Bacteroides/fisiología , Ácidos y Sales Biliares/metabolismo , Fibras de la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/microbiología , Trasplante de Microbiota Fecal , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Pectinas/administración & dosificación , Prebióticos/administración & dosificaciónRESUMEN
The productive human papillomavirus (HPV) life cycle is tightly linked to the differentiation and cycling of keratinocytes. Deregulation of these processes and stimulation of cell proliferation by the action of viral oncoproteins and host cell factors underlies HPV-mediated carcinogenesis. Severe HPV infections characterize the wart, hypogammaglobulinemia, infection, and myelokathexis (WHIM) immunodeficiency syndrome, which is caused by gain-of-function mutations in the CXCR4 receptor for the CXCL12 chemokine, one of which is CXCR41013. We investigated whether CXCR41013 interferes in the HPV18 life cycle in epithelial organotypic cultures. Expression of CXCR41013 promoted stabilization of HPV oncoproteins, thus disturbing cell cycle progression and proliferation at the expense of the ordered expression of the viral genes required for virus production. Conversely, blocking CXCR41013 function restored virus production and limited HPV-induced carcinogenesis. Thus, CXCR4 and its potential activation by genetic alterations in the course of the carcinogenic process can be considered as an important host factor for HPV carcinogenesis.
Asunto(s)
Transformación Celular Viral/fisiología , Quimiocina CXCL12/metabolismo , Infecciones por Papillomavirus/genética , Receptores CXCR4/genética , Transducción de Señal , Neoplasias Cutáneas/virología , Animales , Western Blotting , Línea Celular , Quimiocina CXCL12/genética , Predisposición Genética a la Enfermedad/genética , Xenoinjertos , Papillomavirus Humano 18 , Queratinocitos/metabolismo , Queratinocitos/virología , Ratones , Ratones Desnudos , Receptores CXCR4/metabolismo , Transducción de Señal/fisiología , Neoplasias Cutáneas/genéticaRESUMEN
CXCR4 plays a central role in B cell immune response, notably by promoting plasma cell (PC) migration and maintenance in the bone marrow (BM). Gain-of-function mutations in CXCR4 affecting receptor desensitization have been reported in the rare immunodeficiency called WHIM syndrome (WS). Despite lymphopenia, patients mount an immune response but fail to maintain it over time. Using a knockin mouse model phenocopying WS, we showed that, counter-intuitively, a gain of Cxcr4 function inhibited the maintenance of antibody titers after immunization. Although the Cxcr4 mutation intrinsically and locally promoted germinal center response and PC differentiation, antigen-specific PCs were barely detected in the BM, a defect mirrored by early accumulation of immature plasmablasts potentially occupying the survival niches for long-lived PCs. Therefore, fine-tuning of Cxcr4 desensitization is critically required for efficient PC differentiation and maintenance, and absence of such a regulatory process may account for the defective humoral immunity observed in WS patients.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Médula Ósea/inmunología , Desensibilización Inmunológica , Síndromes de Inmunodeficiencia/inmunología , Células Plasmáticas/inmunología , Receptores CXCR4/inmunología , Verrugas/inmunología , Animales , Anticuerpos/sangre , Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/patología , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Diferenciación Celular , Movimiento Celular , Modelos Animales de Enfermedad , Expresión Génica , Técnicas de Sustitución del Gen , Centro Germinal , Haptenos , Hemocianinas/administración & dosificación , Humanos , Inmunidad Humoral , Inmunización , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Ratones , Ratones Transgénicos , Ovalbúmina/administración & dosificación , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/patología , Enfermedades de Inmunodeficiencia Primaria , Receptores CXCR4/genética , Transducción de Señal , Verrugas/genética , Verrugas/patologíaRESUMEN
Human papillomavirus (HPV) infection is estimated to be the causal agent in 5% of all human cancers and is the leading cause of genital warts, which is the most common sexually transmitted viral disease. Currently, there are no medications to treat HPV infection, and therapeutic strategies primarily target HPV-related cancer rather than viral infection. HPV infection has severe effects on patients who display selective susceptibility to the virus in the context of primary immunodeficiencies, such as the warts, hypogammaglobulinemia, infections, and myelokathexis syndrome, which is caused by dysfunctions of CXCR4, the receptor for the CXCL12 chemokine. In this study we showed in a transgenic mouse model of HPV-induced epidermal neoplasia the beneficial effects of Cxcl12/Cxcr4 pathway blockade with the selective CXCR4 antagonist AMD3100. Daily treatment with AMD3100 for 28 days potently reduced the abnormal ear epidermal thickening in all mice. This effect was associated with reductions in keratinocyte hyperproliferation and immune cell infiltration, both of which are linked to neoplastic progression. Moreover, we observed the abnormal coordinate expression of Cxcl12 and p16INK4a (a surrogate marker of HPV-induced cancers) in dysplastic epidermal keratinocytes, which was inhibited by AMD3100 treatment. These results provide strong evidence for the therapeutic potential of CXCL12/CXCR4 pathway blockade in HPV-induced pathogenesis.
Asunto(s)
Compuestos Heterocíclicos/farmacología , Terapia Molecular Dirigida , Infecciones por Papillomavirus/tratamiento farmacológico , Receptores CXCR4/genética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/virología , Animales , Apoptosis/efectos de los fármacos , Bencilaminas , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclamas , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Queratinocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/genética , Distribución Aleatoria , Receptores CXCR4/efectos de los fármacos , Neoplasias Cutáneas/patologíaRESUMEN
The increase consumption of fructose in diet is associated with liver inflammation. As a specific fructan substrate, fructose may modify the gut microbiota which is involved in obesity-induced liver disease. Here, we aimed to assess whether fructose-induced liver damage was associated with a specific dysbiosis, especially in mice fed a high fat diet (HFD). To this end, four groups of mice were fed with normal and HFD added or not with fructose. Body weight and glucose sensitivity, liver inflammation, dysbiosis and the phenotype of Kupffer cells were determined after 16 weeks of diet. Food intake was increased in the two groups of mice fed with the HFD. Mice fed with HFD and fructose showed a higher infiltration of lymphocytes into the liver and a lower inflammatory profile of Kupffer cells than mice fed with the HFD without fructose. The dysbiosis associated with diets showed that fructose specifically prevented the decrease of Mouse intestinal bacteria in HFD fed mice and increased Erysipelotrichi in mice fed with fructose, independently of the amount of fat. In conclusion, fructose, used as a sweetener, induced a dysbiosis which is different in presence of fat in the diet. Consequently, the activation of Kupffer cells involved in mice model of HFD-induced liver inflammation was not observed in an HFD/fructose combined diet. These data highlight that the complexity of diet composition could highly impact the development of liver lesions during obesity. Specific dysbiosis associated with the diet could explain that the progressions of liver damage are different.
Asunto(s)
Dieta Alta en Grasa , Disbiosis/metabolismo , Fructosa/administración & dosificación , Macrófagos del Hígado/metabolismo , Hígado/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Peso Corporal/efectos de los fármacos , Disbiosis/patología , Ingestión de Alimentos/efectos de los fármacos , Inflamación/metabolismo , Inflamación/patología , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/patología , Hígado/metabolismo , Hígado/patología , Linfocitos/metabolismo , Linfocitos/patología , Ratones , Obesidad/metabolismo , Obesidad/patologíaRESUMEN
BACKGROUND & AIMS: Kupffer cells (KC) play a key role in the onset of inflammation in non-alcoholic steatohepatitis (NASH). The glucocorticoid receptor (GR) induces glucocorticoid-induced leucine zipper (GILZ) expression in monocytes/macrophages and is involved in several inflammatory processes. We hypothesized that the GR-GILZ axis in KC may contribute to the pathophysiology of obesity-induced liver inflammation. METHODS: By using a combination of primary cell culture, pharmacological experiments, mice deficient for the Gr specifically in macrophages and transgenic mice overexpressing Gilz in macrophages, we explored the involvement of the Gr-Gilz axis in KC in the pathophysiology of obesity-induced liver inflammation. RESULTS: Obesity was associated with a downregulation of the Gr and Gilz, and an impairment of Gilz induction by lipopolysaccharide (LPS) and dexamethasone (DEX) in KC. Inhibition of Gilz expression in isolated KC transfected with Gilz siRNA demonstrated that Gilz downregulation was sufficient to sensitize KC to LPS. Conversely, liver inflammation was decreased in obese transgenic mice specifically overexpressing Gilz in macrophages. Pharmacological inhibition of the Gr showed that impairment of Gilz induction in KC by LPS and DEX in obesity was driven by a downregulation of the Gr. In mice specifically deficient for Gr in macrophages, Gilz expression was low, leading to an exacerbation of obesity-induced liver inflammation. CONCLUSIONS: Obesity is associated with a downregulation of the Gr-Gilz axis in KC, which promotes liver inflammation. The Gr-Gilz axis in KC is an important target for the regulation of liver inflammation in obesity.
Asunto(s)
Hepatitis/etiología , Macrófagos del Hígado/fisiología , Obesidad/complicaciones , Receptores de Glucocorticoides/fisiología , Factores de Transcripción/fisiología , Animales , Células Cultivadas , Dexametasona/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones ObesosRESUMEN
Systemic lupus erythematosus is a chronic inflammatory autoimmune disease, the development of which is characterized by a progressive loss of renal function. Such dysfunction is associated with leukocyte infiltration in the glomerular and tubulointerstitial compartments in both human and experimental lupus nephritis. In this study, we investigated the role of the Ccr1 chemokine receptor in this infiltration process during the progression of nephritis in the lupus-prone New Zealand Black/New Zealand White (NZB/W) mouse model. We found that peripheral T cells, mononuclear phagocytes, and neutrophils, but not B cells, from nephritic NZB/W mice were more responsive to Ccr1 ligands than the leukocytes from younger prenephritic NZB/W mice. Short-term treatment of nephritic NZB/W mice with the orally available Ccr1 antagonist BL5923 decreased renal infiltration by T cells and macrophages. Longer Ccr1 blockade decreased kidney accumulation of effector/memory CD4(+) T cells, Ly6C(+) monocytes, and both M1 and M2 macrophages; reduced tubulointerstitial and glomerular injuries; delayed fatal proteinuria; and prolonged animal lifespan. In contrast, renal humoral immunity was unaffected in BL5923-treated mice, which reflected the unchanged numbers of infiltrated B cells in the kidneys. Altogether, these findings define a pivotal role for Ccr1 in the recruitment of T and mononuclear phagocyte cells to inflamed kidneys of NZB/W mice, which in turn contribute to the progression of renal injury.
Asunto(s)
Nefritis Lúpica/terapia , Células Mieloides/inmunología , Infiltración Neutrófila , Receptores CCR1/antagonistas & inhibidores , Subgrupos de Linfocitos T/inmunología , Factores de Edad , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Quimiocina CCL3/biosíntesis , Quimiocina CCL3/deficiencia , Quimiocina CCL3/genética , Quimiocina CCL3/fisiología , Quimiocina CCL5/biosíntesis , Quimiocina CCL5/genética , Quimiocina CCL5/fisiología , Quimiotaxis de Leucocito , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Riñón/inmunología , Riñón/patología , Ligandos , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Ratones , Ratones Endogámicos NZB , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Células Mieloides/patología , Infiltración Neutrófila/efectos de los fármacos , ARN Mensajero/biosíntesis , Distribución Aleatoria , Receptores CCR1/biosíntesis , Receptores CCR1/genética , Receptores CCR1/fisiología , Bazo/inmunología , Bazo/patología , Esplenomegalia/etiología , Esplenomegalia/inmunología , Esplenomegalia/patología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patologíaRESUMEN
Ovarian cancer is the gynecological cancer exhibiting the highest morbidity and improvement of treatments is still required. Previous studies have shown that Estrogen-receptor beta (ERß) levels decreased along with ovarian carcinogenesis. Here, we present evidence that reintroduction of ERß in BG-1 epithelial ovarian cancer cells, which express ERα, leads in vitro to a decrease of basal and estradiol-promoted cell proliferation. ERß reduced the frequency of cells in S phase and increased the one of cells in G2/M phase. At the molecular level, we found that ERß downregulated total retinoblastoma (Rb), phosphorylated Rb and phospho-AKT cellular content as well as cyclins D1 and A2. In addition, ERß had a direct effect on ERα, by strongly inhibiting its expression and activity, which could explain part of the anti-proliferative action of ERß. By developing a novel preclinical model of ovarian cancer based on a luminescent orthotopic xenograft in athymic Nude mice, we further revealed that ERß expression reduces tumor growth and the presence of tumor cells in sites of metastasis, hence resulting in improved survival of mice. Altogether, these findings unveil a potential tumor-suppressor role of ERß in ovarian carcinogenesis, which could be of potential clinical relevance for the selection of the most appropriate treatment for patients.
Asunto(s)
Receptor beta de Estrógeno/fisiología , Genes Supresores de Tumor , Neoplasias Glandulares y Epiteliales/fisiopatología , Neoplasias Ováricas/fisiopatología , Proliferación Celular , Receptor beta de Estrógeno/genética , Femenino , Humanos , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Fosforilación , Proteína de Retinoblastoma/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is characterized by steatosis associated with liver inflammation. Steatosis causes recruitment of lymphocytes into the liver and this is worsened by lipopolysaccharides (LPS). As macrophages may be involved in the lymphocyte homing, we studied the role of lipids in determining the phenotype of Kupffer cells (KCs) at the stage of steatosis. METHODS: Steatosis was induced in mice by a high fat diet. The turnover and the recruitment of KCs were analyzed in vivo by flow cytometry. KCs phenotype was assessed by optical and electron microscopy, cell culture and lymphocyte recruitment by in vitro chemotaxis. Lipidomic analysis was carried out by mass-spectrometry and gene expression analysis by TaqMan low density array. RESULTS: Although the number of KCs was not modified in steatotic livers compared to normal livers, their phenotypes were different. Electron microscopy demonstrated that the KCs from fatty livers were enlarged and loaded with lipid droplets. Lipid synthesis and trafficking were dysregulated in fat-laden KCs and toxic lipids accumulated. Fat-laden KCs recruited more CD4+ T and B lymphocytes in response to LPS stimulation than did control KCs and produced high levels of pro-inflammatory cytokines/chemokines, which could be reversed by inhibition of lipogenesis. CONCLUSIONS: Lipid accumulation in fat-laden KCs is due to a dysregulation of lipid metabolism and trafficking. Fat-laden KCs are "primed" to recruit lymphocytes and exhibit a pro-inflammatory phenotype, which is reversible with inhibition of lipogenesis.
Asunto(s)
Hígado Graso/inmunología , Hígado Graso/metabolismo , Macrófagos del Hígado/inmunología , Macrófagos del Hígado/metabolismo , Acetiltransferasas/genética , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Carnitina O-Palmitoiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/genética , Grasas de la Dieta/metabolismo , Grasas de la Dieta/toxicidad , Ácido Graso Sintasas/genética , Proteínas de Unión a Ácidos Grasos/genética , Hígado Graso/patología , Expresión Génica/fisiología , Macrófagos del Hígado/patología , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Enfermedad del Hígado Graso no Alcohólico , Proteínas Nucleares/genética , Obesidad/inmunología , Obesidad/metabolismo , PPAR gamma/genética , Fenotipo , Estearoil-CoA Desaturasa/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Little is known about the molecules that contribute to the growth of epithelial ovarian carcinomas (EOC), which remain the most lethal gynecological cancer in women. The chemokine Fractalkine/CX(3)CL1 has been widely reported to play a biologically relevant role in tumor growth and spread. We report here the first investigation of the expression and role of CX(3)CL1 in EOC. RESULTS: Epithelial cells from the surface of the ovary and the Fallopian tubes and from benign, borderline and malignant tumors all stained positive for CX(3)CL1. In tumor specimens from 54 women who underwent surgical treatment for EOC diagnosis, CX(3)CL1 immunoreactivity was unevenly distributed in epithelial tumor cells, and ranged from strong (33%) to absent (17%). This uneven distribution of CX(3)CL1 did not reflect the morphological heterogeneity of EOC. It was positively correlated with the proliferation index Ki-67 and with GILZ (glucocorticoid-induced leucine zipper), previously identified as an activator of the proliferation of malignant EOC cells. Hierarchical clustering analysis, including age at diagnosis, tumor grade, FIGO stage, Ki-67 index, CX(3)CL1, SDF-1/CXCL12 and GILZ immunostaining scores, distinguished two major clusters corresponding to low and high levels of proliferation and differing in terms of GILZ and CX(3)CL1 expression. GILZ overexpression in the carcinoma-derived BG1 cell line resulted in parallel changes in CX(3)CL1 products. Conversely, CX(3)CL1 promoted through its binding to CX(3)CR1 AKT activation and proliferation in BG1 cells. In a mouse subcutaneous xenograft model, the overexpression of GILZ was associated with higher expression of CX(3)CL1 and faster tumor growth. CONCLUSION: Our findings highlight the previously unappreciated constitutive expression of CX(3)CL1 preceding tumorigenesis in ovarian epithelial cells. Together with GILZ, this chemokine emerges as a regulator of cell proliferation, which may be of potential clinical relevance for the selection of the most appropriate treatment for EOC patients.
Asunto(s)
Proliferación Celular , Quimiocina CX3CL1/metabolismo , Células Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Western Blotting , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Quimiocina CX3CL1/genética , Células Epiteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante Heterólogo , Carga TumoralRESUMEN
BACKGROUND: CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value. METHODS: Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves. RESULTS: Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases (<10%). This uneven distribution of CXCL12 did not reflect the morphological heterogeneity of EOC. CXCL12 expression levels were not correlated with any of the clinical parameters currently used to determine EOC prognosis or with HER2 status. They also had no impact on progression-free or overall survival. CONCLUSION: Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant epithelial cells precedes tumorigenesis and we confirm in a large cohort of patients with advanced EOC that CXCL12 expression level in EOC is not a valuable prognostic factor in itself. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00052468.
Asunto(s)
Quimiocina CXCL12/metabolismo , Células Epiteliales/metabolismo , Ovario/metabolismo , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Carcinoma Epitelial de Ovario , Ensayos Clínicos Fase III como Asunto/estadística & datos numéricos , Estudios de Cohortes , Células Epiteliales/patología , Femenino , Salud , Humanos , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/diagnóstico , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Ovario/patología , Valor Predictivo de las Pruebas , Ensayos Clínicos Controlados Aleatorios como Asunto/estadística & datos numéricosRESUMEN
BACKGROUND: Little is known about the molecules that contribute to tumor progression of epithelial ovarian cancer (EOC), currently a leading cause of mortality from gynecological malignancies. Glucocorticoid-Induced Leucine Zipper (GILZ), an intracellular protein widely expressed in immune tissues, has been reported in epithelial tissues and controls some of key signaling pathways involved in tumorigenesis. However, there has been no report on GILZ in EOC up to now. The objectives of the current study were to examine the expression of GILZ in EOC and its effect on tumor cell proliferation. RESULTS: GILZ expression was measured by immunohistochemical staining in tissue sections from 3 normal ovaries, 7 benign EOC and 50 invasive EOC. GILZ was not detected on the surface epithelium of normal ovaries and benign tumors. In contrast, it was expressed in the cytoplasm of tumor cells in 80% EOC specimens. GILZ immunostaining scores correlated positively to the proliferation marker Ki-67 (Spearman test in univariate analysis, P < 0.00001, r = 0.56). They were also higher in tumor cells containing large amounts of phosphorylated protein kinase B (p-AKT) (unpaired t test, P < 0.0001). To assess the effect of GILZ on proliferation and AKT activation, we used the BG-1 cell line derived from ovarian tumor cells as a cellular model. GILZ expression was either enhanced by stable transfection or decreased by the use of small interfering (si) RNA targeting GILZ. We found that GILZ increased cell proliferation, phospho-AKT cellular content and AKT kinase activity. Further, GILZ upregulated cyclin D1 and phosphorylated retinoblastoma (p-Rb), downregulated cyclin-dependent kinase inhibitor p21, and promoted the entry into S phase of cell cycle. CONCLUSION: The present study is the first to identify GILZ as a molecule produced by ovarian cancer cells that promotes cell cycle progression and proliferation. Our findings clearly indicate that GILZ activates AKT, a crucial signaling molecule in tumorigenesis. GILZ thus appears as a potential key molecule in EOC.
Asunto(s)
Células Epiteliales/patología , Neoplasias Ováricas/patología , Factores de Transcripción/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proliferación Celular , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Activación Enzimática , Células Epiteliales/enzimología , Femenino , Silenciador del Gen , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/enzimología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of long-lived monoclonal B cells mostly arrested at the G(0)/G(1) phase of the cell cycle. CLL cells strongly express intracellular melanoma differentiation-associated gene-7 (MDA7)/IL-24. However, adenovirus-delivered MDA7 was reported to be cytotoxic in several tumor cell lines. We report herein that rIL-24 alone had no effect; however, sequential incubation with rIL-2 and rIL-24 reduced thymidine incorporation by 50% and induced apoptosis of CLL cells in S and G(2)/M phases of the cell cycle, but not of normal adult blood or tonsil B cells. IL-24 stimulated STAT3 phosphorylation in IL-24R1-transfected cells but not in normal or CLL B cells. In contrast, IL-24 reversed the IL-2-induced phosphorylation of STAT3 in CLL, and this effect was neutralized by anti-IL-24 Ab. Phospho- (P)STAT3 inhibition induced by IL-24 was reversed by pervanadate, an inhibitor of tyrosine phosphatases. The addition of rIL-24 to IL-2-activated CLL B cells resulted in increases of transcription, protein synthesis. and phosphorylation of p53. The biological effects of IL-24 were reversed by the p53 inhibitor pifithrin-alpha and partly by the caspase inhibitor zvad. Troglitazone (a protein tyrosine phosphatase, PTP1B activator) phosphatase inhibited PSTAT3 and augmented p53 expression. PSTAT3 is a transcriptional repressor of p53, and therefore IL-24 induction of p53 secondary to PSTAT3 dephosphorylation may be sensed as a stress signal and promote apoptosis in cycling cells. This model explains why IL-24 can protect some resting/differentiated cells and be deleterious to proliferating cells.