RESUMEN
Despite extensive investigations, an effective treatment for sepsis remains elusive and a better understanding of the inflammatory response to infection is required to identify potential new targets for therapy. In this study we have used RNAi technology to show, for the first time, that the inducible lysophosphatidylcholine acyltransferase 2 (LPCAT2) plays a key role in macrophage inflammatory gene expression in response to stimulation with bacterial ligands. Using siRNA- or shRNA-mediated knockdown, we demonstrate that, in contrast to the constitutive LPCAT1, LPCAT2 is required for macrophage cytokine gene expression and release in response to TLR4 and TLR2 ligand stimulation but not for TLR-independent stimuli. In addition, cells transfected to overexpress LPCAT2 exhibited increased expression of inflammatory genes in response to LPS and other bacterial ligands. Furthermore, we have used immunoprecipitation and Western blotting to show that in response to LPS, LPCAT2, but not LPCAT1, rapidly associates with TLR4 and translocates to membrane lipid raft domains. Our data thus suggest a novel mechanism for the regulation of inflammatory gene expression in response to bacterial stimuli and highlight LPCAT2 as a potential therapeutic target for development of anti-inflammatory and anti-sepsis therapies.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Macrófagos Peritoneales/inmunología , Monocitos/inmunología , Sepsis/inmunología , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , Animales , Línea Celular Tumoral , Regulación de la Expresión Génica/inmunología , Técnicas de Silenciamiento del Gen , Humanos , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/metabolismo , Microdominios de Membrana/metabolismo , Ratones , Monocitos/citología , Monocitos/metabolismo , Cultivo Primario de Células , Células RAW 264.7 , ARN Interferente Pequeño/metabolismo , Sepsis/microbiología , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 4/metabolismoRESUMEN
The major phospholipid in pulmonary surfactant dipalmitoyl phosphatidylcholine (DPPC) has been shown to modulate inflammatory responses. Using human monocytes, this study demonstrates that DPPC significantly increased PGE(2) (P<0.05) production by 2.5-fold when compared to untreated monocyte controls. Mechanistically, this effect was concomitant with an increase in COX-2 expression which was abrogated in the presence of a COX-2 inhibitor. The regulation of COX-2 expression was independent of NF-kappaB activity. Further, DPPC increased the phosphorylation of the cyclic AMP response element binding protein (CREB; an important nuclear transcription factor important in regulating COX-2 expression). In addition, we also show that changing the fatty acid groups of PC (e.g. using l-alpha-phosphatidylcholine beta-arachidonoyl-gamma-palmitoyl (PAPC)) has a profound effect on the regulation of COX-2 expression and CREB activation. This study provides new evidence for the anti-inflammatory activity of DPPC and that this activity is at least in part mediated via CREB activation of COX-2.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Ciclooxigenasa 2/metabolismo , Monocitos/enzimología , Surfactantes Pulmonares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/farmacología , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclooxigenasa 2/genética , Dinoprostona/biosíntesis , Dinoprostona/metabolismo , Humanos , Monocitos/efectos de los fármacos , Fosforilación , Surfactantes Pulmonares/química , Surfactantes Pulmonares/farmacologíaRESUMEN
Honey is used as a therapy to aid wound healing. Previous data indicate that honey can stimulate cytokine production from human monocytes. The present study further examines this phenomenon in manuka honey. As inflammatory cytokine production in innate immune cells is classically mediated by pattern recognition receptors in response to microorganisms, bacterial contamination of honey and the effect of blocking TLR2 and -4 on stimulatory activity were assessed. No vegetative bacteria were isolated from honey; however, bacterial spores were cultured from one-third of samples, and low levels of LPS were detected. Blocking TLR4 but not TLR2 inhibited honey-stimulated cytokine production significantly. Cytokine production did not correlate with LPS levels in honey and was not inhibited by polymyxin B. Further, the activity was reduced significantly following heat treatment, indicating that component(s) other than LPS are responsible for the stimulatory activity of manuka honey. To identify the component responsible for inducing cytokine production, honey was separated by molecular weight using microcon centrifugal filtration and fractions assessed for stimulatory activity. The active fraction was analyzed by MALDI-TOF mass spectroscopy, which demonstrated the presence of a number of components of varying molecular weights. Additional fractionation using miniaturized, reverse-phase solid-phase extraction resulted in the isolation of a 5.8-kDa component, which stimulated production of TNF-alpha via TLR4. These findings reveal mechanisms and components involved in honey stimulation of cytokine induction and could potentially lead to the development of novel therapeutics to improve wound healing for patients with acute and chronic wounds.
Asunto(s)
Miel , Leptospermum , Monocitos/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Médula Ósea/metabolismo , Humanos , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Monocitos/citología , Monocitos/inmunología , Polimixina B/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/inmunología , Staphylococcus aureus/metabolismo , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The t(8;21) translocation is one of the most frequent translocations in acute myeloid leukaemia (AML), giving rise to the AML1-ETO fusion protein (or RUNX1-CBF2T1). This abnormality is associated with myelocytic leukaemia with dysplastic granulopoiesis. Here, we demonstrate that when expressed in a normal human (CD34(+)) progenitor population, AML1-ETO selectively inhibits granulocyte colony formation but not monocyte colony formation. In bulk liquid culture, we found that though AML1-ETO transiently inhibited the proliferation of CD34(+) cells, it promoted long-term growth of myeloid cells for more than 80 days, suggesting that differentiation was inhibited. In support of this, cultures expressing AML1-ETO demonstrated enhanced retention of colony-forming capacity. Phenotypic examination of AML1-ETO cultures revealed a defect in granulocytic differentiation in terms of retention of CD34(+) cells within the culture and delayed CD11b upregulation. Morphologically, granulocyte terminal differentiation in AML1-ETO-expressing cells was inhibited by 83+/-5%, giving rise to a build-up of early to intermediate granulocytes that exhibited a number of morphological features associated with t(8;21) leukaemias. In contrast, AML1-ETO had little or no effect on monocytic differentiation. Taken together, these results suggest that expression of AML1-ETO selectively inhibits the differentiation of granulocytic cells and promoted extensive self-renewal, supporting a causal role for t(8;21) translocations in leukaemogenesis.
Asunto(s)
Células Precursoras de Granulocitos/patología , Leucemia Mielomonocítica Aguda/patología , Proteínas de Fusión Oncogénica/fisiología , Factores de Transcripción/fisiología , Antígenos CD34 , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , División Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Células Eritroides/patología , Proteínas Fluorescentes Verdes , Humanos , Inmunofenotipificación , Leucemia Mielomonocítica Aguda/etiología , Proteínas Luminiscentes/genética , Células Mieloides/patología , Proteínas de Fusión Oncogénica/genética , Proteína 1 Compañera de Translocación de RUNX1 , Factores de Tiempo , Factores de Transcripción/genética , Transducción GenéticaRESUMEN
Clinical observations indicate that honey may initiate or accelerate the healing of chronic wounds and has, therefore, been claimed to have anti-inflammatory properties. The aim of this study was to investigate the effects of honey on the activation state of immunocompetent cells, using the monocytic cell line, MonoMac-6 (MM6), as a model. We investigated the effect of each of the three honeys (manuka, pasture and jelly bush) on the release of important inflammatory cytokines from MM6 cells. These honeys, together with a sugar syrup control (artificial honey), were incubated with MM6 cells at a concentration of 1% (w/v) for 0-24h. Cell culture supernatants were tested using specific ELISA assays for tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta and IL-6. All honeys significantly increased the TNF-alpha, IL-1beta and IL-6 release from MM6 cells (and human monocytes) when compared with untreated and artificial-honey-treated cells (P<0.001). Jelly bush honey significantly induced the maximal release of each cytokine compared with manuka, pasture or artificial honeys (P<0.001). These results suggest that the effect of honey on wound healing may in part be related to the stimulation of inflammatory cytokines from monocytic cells. Such cell types are known to play an important role in healing and tissue repair.