RESUMEN
Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.
Asunto(s)
Toxinas Bacterianas/antagonistas & inhibidores , Células Epiteliales/efectos de los fármacos , Proteínas Hemolisinas/antagonistas & inhibidores , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inmunidad Innata/efectos de los fármacos , Simvastatina/farmacología , Estreptolisinas/antagonistas & inhibidores , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Línea Celular Transformada , Células Epiteliales/inmunología , Células Epiteliales/patología , Proteínas Hemolisinas/toxicidad , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/inmunología , Inyecciones Intraperitoneales , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Pravastatina/inmunología , Pravastatina/farmacología , Cultivo Primario de Células , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Simvastatina/inmunología , Staphylococcus aureus/química , Streptococcus pneumoniae/química , Estreptolisinas/toxicidadRESUMEN
Intestinal ischemia/reperfusion (I/R) injury is a grave condition with high morbidity and mortality. We previously confirmed that intestinal I/R induces intestinal flora disorders and changes in metabolites, but the role of different metabolites in intestinal I/R injury is currently unclear. Based on targeted metabolic sequencing, pravastatin (PA) was determined to be a metabolite of the gut microbiota. Further, intestinal I/R model mice were established through superior mesenteric artery obstruction. In addition, a co-culture model of small intestinal organoids and type II innate lymphoid cells (ILC2s) was subjected to hypoxia/reoxygenation (H/R) to simulate an intestinal I/R model. Moreover, correlation analysis between the PA level in preoperative feces of patients undergoing cardiopulmonary bypass and the indices of postoperative intestinal I/R injury was carried out. IL-33-deficient mice, ILC2-deleted mice, and anti-IL-13 neutralizing antibodies were also used to explore the potential mechanism through which PA attenuates intestinal I/R injury. We demonstrated that PA levels in the preoperative stool of patients undergoing cardiopulmonary bypass were negatively correlated with the indices of postoperative intestinal I/R injury. Furthermore, PA alleviated intestinal I/R injury and improved the survival of mice. We further showed that PA promotes IL-13 release from ILC2s by activating IL-33/ST2 signaling to attenuate intestinal I/R injury. In addition, IL-13 promoted the self-renewal of intestinal stem cells by activating Notch1 and Wnt signals. Overall, results indicated that the gut microbial metabolite PA can attenuate intestinal I/R injury by promoting the release of IL-13 from ILC2s via IL-33/ST2 signaling, revealing a novel mechanism of and therapeutic strategy for intestinal I/R injury.
Asunto(s)
Microbioma Gastrointestinal/inmunología , Inmunidad Innata , Proteína 1 Similar al Receptor de Interleucina-1/inmunología , Interleucina-13/inmunología , Interleucina-33/inmunología , Enfermedades Intestinales/inmunología , Linfocitos/inmunología , Pravastatina/inmunología , Animales , Modelos Animales de Enfermedad , Humanos , Proteína 1 Similar al Receptor de Interleucina-1/genética , Interleucina-13/genética , Interleucina-33/genética , Enfermedades Intestinales/genética , Masculino , Ratones , Ratones Noqueados , Daño por ReperfusiónRESUMEN
OBJECTIVE: MHC class II molecules play central roles in immune recognition and rejection. As statins have been shown to inhibit the production of these molecules, we analyzed the possible immunosuppressive effect of pravastatin, using our rat model of orthotopic lung transplantation. METHODS: Single orthotopic lung transplantation was performed in a Fischer 344-to-Wistar Kyoto strain combination. One group received pravastatin i.p. after transplantation, controls NaCl. Statin serum levels were analyzed by high performance liquid chromatography (HPLC). Animals were sacrificed on postoperative day (POD) 14 and 21. At sacrifice, samples were obtained for histology, immunhistochemistry, flow cytometry and real-time RT-PCR analysis of CD25, TNF-alpha, and MHC class II expression. Rejection was graded via histochemistry, using a system based on the working formulation of The International Society of Heart and Lung Transplantation. Immunohistochemistry was performed for expression of MHC class II, T-cell receptor, CD25, CD4/8 cell, NK cells, granulocytes and monocytes in naïve lungs and grafts from donor and recipient animals. Flow cytometric analysis of recipient peripheral blood mononuclear cells (PBMC) was used to analyze expression of CD3, CD4, CD8, and RT1B in both groups. In vitro analyses of MHC class II expression were performed in parallel. RESULTS: HPLC confirmed effective delivery of pravastatin. Recipients treated with pravastatin showed significantly less rejection on POD 14 and on POD 21, when compared to controls. Immunohistochemistry showed specific differences, suggesting a delay in rejection in the pravastatin group. Flow cytometric analyses showed a higher expression of CD4 in the control group on POD 21. Results of real-time RT-PCR analyses for MHC class II expression showed a significant decrease in expression in the statin-treated group. Flow cytometric analysis of gamma-IFN stimulated rat PBMC showed an inhibition of upregulation of MHC class II expression by pravastatin in vitro. CONCLUSIONS: Pravastatin prolongs graft survival in our allogeneic rat model of orthotopic lung transplantation. We assume that the underlying mechanism for this effect is the inhibition of upregulation of MHC class II molecule synthesis, thus blocking downstream effector mechanisms of the immune system.
Asunto(s)
Supervivencia de Injerto/inmunología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/inmunología , Trasplante de Pulmón/métodos , Pravastatina/inmunología , Animales , Bronquitis/inmunología , Antígenos CD4/inmunología , Citometría de Flujo/métodos , Rechazo de Injerto/inmunología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/sangre , Tolerancia Inmunológica/inmunología , Inmunohistoquímica/métodos , Leucocitos Mononucleares/inmunología , Masculino , Modelos Animales , Pravastatina/sangre , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas WKY , Receptores de Interleucina-2/análisis , Receptores de Interleucina-2/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
A method for the determination of pravastatin sodium (PS), a cholesterol-lowering agent, in plasma was developed by using an immobilized antibody column extraction followed by high-performance liquid chromatography (HPLC). The analyte was monitored by a laser-induced fluorescence detector after fluorogenic derivatization. The PS antibody was coupled to Sepharose 4B and used as an extraction phase for sample cleanup and extraction of the drug. A plasma sample was applied to the column and washed with water, and the drug was eluted with methanol. N-Dansylethylenediamine (DNS-ED) was coupled to the carboxyl moiety of the drug in the presence of diethyl phosphorocyanidate (DEPC) and triethylamine (TEA) in dioxane. Derivatization was completed in 5 min at room temperature. A column-switching technique was utilized to remove excess reagents and byproducts. A He-Cd laser-induced fluorescence detector was applied to achieve an ultrasensitive determination. The detection limit was 2 pg/injection of PS, which was 20 times more sensitive than the conventional fluorescence detection. The limit of quantitation was 100 pg/mL when 1 mL of plasma sample was available. An average coefficient of variations of the overall method were less than 8% at the concentration range of 1-100 ng/mL. A single oral dose of PS in rats (20 mg/kg) and dogs (5 mg/kg) resulted in average maximum concentrations of 142 and 310 ng/mL, respectively.
Asunto(s)
Pravastatina/sangre , Animales , Anticuerpos/análisis , Anticuerpos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Perros , Ensayo de Inmunoadsorción Enzimática , Indicadores y Reactivos , Rayos Láser , Masculino , Factor de Activación Plaquetaria/inmunología , Factor de Activación Plaquetaria/aislamiento & purificación , Pravastatina/inmunología , Pravastatina/farmacocinética , Ratas , Ratas Wistar , Espectrometría de FluorescenciaRESUMEN
Statin drugs prevent coronary heart disease through anti-inflammatory mechanisms in addition to the well-known reduction of low-density lipoproteins. The complement system plays an essential role in the inflammatory response and has been postulated to be modified by statins. A direct role for statins in complement activation, however, has not been previously investigated. We therefore studied the effect of statins on in vitro complement activation. Pravastatin, atorvastatin and the active metabolite of the latter, ortho-hydroxy atorvastatin, were added to normal human serum and incubated for 1 h in the absence or presence of aggregated immunoglobulin (classical pathway activation) or cobra venom factor (alternative pathway activation). The degree of complement activation, as detected by specific complement-activation products for the classical pathway (C1rs-C1-inhibitor complexes), the combined classical and lectin pathway (C4bc), the alternative pathway (C3bBbP) and the final common pathway (C3bc and TCC), was not affected by pre-incubation of the serum with any of the statins. Statins do not affect complement activation directly, but indirect effects in vivo may well be operative.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/efectos de los fármacos , Ácidos Heptanoicos/farmacología , Pravastatina/farmacología , Pirroles/farmacología , Atorvastatina , Activación de Complemento/inmunología , Vía Alternativa del Complemento/efectos de los fármacos , Vía Alternativa del Complemento/inmunología , Vía Clásica del Complemento/efectos de los fármacos , Vía Clásica del Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Venenos Elapídicos/inmunología , Ácidos Heptanoicos/inmunología , Humanos , Inmunoglobulinas/inmunología , Pravastatina/inmunología , Pirroles/inmunologíaRESUMEN
We previously reported that human interleukin (IL)-2 dependent T cell lines derived from very late antigen (VLA)-1(+) CD45RO(+) peripheral blood (PB) T-cells adhere constitutively to collagen type IV, whereas lines from VLA-1(-) PB lymphocytes (L) adhere weakly. Here we report that the latter are induced to adhere by phorbol 12-myristate 13-acetate (PMA). Both PMA dependent and constitutive adhesion, including that of a Herpes Virus Saimiri (HVS) infected CD4(+)VLA-1(+) clone (HVST) were inhibited by anti-VLA-1 monoclonal antibodies (mAb), by inhibitors of phospholipase C (PLC)gamma and by lovastatin but not by a MEK1 inhibitor, whereas only PMA induced adhesion was blocked by inhibition of protein-kinase (PK) C. Furthermore, lovastatin enhanced PLCgamma and anti VLA-1 mAb blockade, and its effect was not reversed by mevalonic acid (MVA). Lovastatin also inhibited interferon (IFN)gamma secretion by T cells triggered with anti-CD3 and in cells detaching from collagen IV. These results suggest new ways for functional modulation of activated T-cells interacting with collagen.