RESUMEN
Dynamic regulation of the mitochondrial network by mitofusins (MFNs) modulates energy production, cell survival, and many intracellular signaling events, including calcium handling. However, the relative importance of specific mitochondrial functions and their dependence on MFNs vary greatly among cell types. Osteoclasts have many mitochondria, and increased mitochondrial biogenesis and oxidative phosphorylation enhance bone resorption, but little is known about the mitochondrial network or MFNs in osteoclasts. Because expression of each MFN isoform increases with osteoclastogenesis, we conditionally deleted MFN1 and MFN2 (double conditional KO (dcKO)) in murine osteoclast precursors, finding that this increased bone mass in young female mice and abolished osteoclast precursor differentiation into mature osteoclasts in vitro Defective osteoclastogenesis was reversed by overexpression of MFN2 but not MFN1; therefore, we generated mice lacking only MFN2 in osteoclasts. MFN2-deficient female mice had increased bone mass at 1 year and resistance to Receptor Activator of NF-κB Ligand (RANKL)-induced osteolysis at 8 weeks. To explore whether MFN-mediated tethering or mitophagy is important for osteoclastogenesis, we overexpressed MFN2 variants defective in either function in dcKO precursors and found that, although mitophagy was dispensable for differentiation, tethering was required. Because the master osteoclastogenic transcriptional regulator nuclear factor of activated T cells 1 (NFATc1) is calcium-regulated, we assessed calcium release from the endoplasmic reticulum and store-operated calcium entry and found that the latter was blunted in dcKO cells. Restored osteoclast differentiation by expression of intact MFN2 or the mitophagy-defective variant was associated with normalization of store-operated calcium entry and NFATc1 levels, indicating that MFN2 controls mitochondrion-endoplasmic reticulum tethering in osteoclasts.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Diferenciación Celular , GTP Fosfohidrolasas/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Animales , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/genética , Ratones , Ratones Noqueados , Mitofagia , Factores de Transcripción NFATC/genética , Osteoclastos/citologíaRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent multidrug-resistant pathogens worldwide, exhibiting increasing resistance to the latest antibiotic therapies. Here we show that the triple ß-lactam combination meropenem-piperacillin-tazobactam (ME/PI/TZ) acts synergistically and is bactericidal against MRSA subspecies N315 and 72 other clinical MRSA isolates in vitro and clears MRSA N315 infection in a mouse model. ME/PI/TZ suppresses evolution of resistance in MRSA via reciprocal collateral sensitivity of its constituents. We demonstrate that these activities also extend to other carbapenem-penicillin-ß-lactamase inhibitor combinations. ME/PI/TZ circumvents the tight regulation of the mec and bla operons in MRSA, the basis for inducible resistance to ß-lactam antibiotics. Furthermore, ME/PI/TZ subverts the function of penicillin-binding protein-2a (PBP2a) via allostery, which we propose as the mechanism for both synergy and collateral sensitivity. Showing in vivo activity similar to that of linezolid, ME/PI/TZ demonstrates that combinations of older ß-lactam antibiotics could be effective against MRSA infections in humans.
Asunto(s)
Antibacterianos/farmacología , Resistencia a la Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , Inhibidores de beta-Lactamasas/farmacología , Regulación Alostérica , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Expresión Génica , Humanos , Linezolid/farmacología , Meropenem , Resistencia a la Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Ratones , Ratones Endogámicos ICR , Pruebas de Sensibilidad Microbiana , Operón , Ácido Penicilánico/análogos & derivados , Ácido Penicilánico/farmacología , Proteínas de Unión a las Penicilinas , Piperacilina/farmacología , Infecciones Estafilocócicas/microbiología , Tazobactam , Tienamicinas/farmacología , beta-Lactamasas/química , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila. Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.
Asunto(s)
Membrana Celular/metabolismo , Extensiones de la Superficie Celular/metabolismo , Uniones Intercelulares/metabolismo , Tetrahymena thermophila/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/fisiología , Espacio Extracelular/metabolismo , Uniones Intercelulares/ultraestructura , Metabolismo de los Lípidos , Vesículas Secretoras/metabolismo , Tetrahymena thermophila/fisiología , Tetrahymena thermophila/ultraestructuraRESUMEN
Mitochondria are essential organelles that form highly complex, interconnected dynamic networks inside cells. The GTPase mitofusin 2 (MFN2) is a highly conserved outer mitochondrial membrane protein involved in the regulation of mitochondrial morphology, which can affect various metabolic and signaling functions. The role of mitochondria in bone formation remains unclear. Since MFN2 levels increase during osteoblast (OB) differentiation, we investigated the role of MFN2 in the osteolineage by crossing mice bearing floxed Mfn2 alleles with those bearing Prx-cre to generate cohorts of conditional knock out (cKO) animals. By ex vivo microCT, cKO female mice, but not males, display an increase in cortical thickness at 8, 18, and 30 weeks, compared to wild-type (WT) littermate controls. However, the cortical anabolic response to mechanical loading was not different between genotypes. To address how Mfn2 deficiency affects OB differentiation, bone marrow-derived mesenchymal stromal cells (MSCs) from both wild-type and cKO mice were cultured in osteogenic media with different levels of ß-glycerophosphate. cKO MSCs show increased mineralization and expression of multiple markers of OB differentiation only at the lower dose. Interestingly, despite showing the expected mitochondrial rounding and fragmentation due to loss of MFN2, cKO MSCs have an increase in oxygen consumption during the first 7 days of OB differentiation. Thus, in the early phases of osteogenesis, MFN2 restrains oxygen consumption thereby limiting differentiation and cortical bone accrual during homeostasis in vivo.
Asunto(s)
GTP Fosfohidrolasas , Osteogénesis , Animales , Diferenciación Celular , Hueso Cortical/diagnóstico por imagen , Femenino , GTP Fosfohidrolasas/genética , Ratones , Ratones NoqueadosRESUMEN
Alternative NF-κB signaling promotes osteoclastogenesis and pathological bone loss, but the effect of sex on phenotype has not been explored. We disrupted alternative NF-κB signaling by deletion of upstream kinase NF-κB-inducing kinase (NIK) or NF-κB subunit RelB and found that both NIK-deficient and RelB-deficient female mice possessed more than twofold higher trabecular bone mass compared to controls, whereas no differences were observed in males. In vitro, RelB-deficient precursors from female mice showed a more severe osteoclast (OC) differentiation defect than male, while WT had no sex bias. Next, we asked whether pharmacologic activation of alternative NF-κB by inhibitor of apoptosis (IAP) antagonist BV6 has sex-dependent effects on bone. Unlike male mice that lost bone, female mice on BV6 for 4 weeks showed no changes in either trabecular bone mass or OC number. Because estrogen generally suppresses NF-κB, we hypothesized that estrogen protects bone from BV6 effects in vivo. Thus, we performed ovariectomy or sham surgery in female mice, then treated with BV6 or vehicle for 4 weeks. Although ovariectomy caused bone loss, BV6 did not have any additional impact, suggesting that direct estrogen effects do not cause resistance to BV6 in vivo. The osteopenic effects of IAP antagonists in males may have implications for their use in cancer therapy. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
RESUMEN
Osteomyelitis (OM), or inflammation of bone tissue, occurs most frequently as a result of bacterial infection and severely perturbs bone structure. OM is predominantly caused by Staphylococcus aureus, and even with proper treatment, OM has a high rate of recurrence and chronicity. While S. aureus has been shown to infect osteoblasts, it remains unclear whether osteoclasts (OCs) are also a target of intracellular infection. Here, we demonstrate the ability of S. aureus to intracellularly infect and divide within OCs. OCs were differentiated from bone marrow macrophages (BMMs) by exposure to receptor activator of nuclear factor kappa-B ligand (RANKL). By utilizing an intracellular survival assay and flow cytometry, we found that at 18 h postinfection the intracellular burden of S. aureus increased dramatically in cells with at least 2 days of RANKL exposure, while the bacterial burden decreased in BMMs. To further explore the signals downstream of RANKL, we manipulated factors controlling OC differentiation, NFATc1 and alternative NF-κB, and found that intracellular bacterial growth correlates with NFATc1 levels in RANKL-treated cells. Confocal and time-lapse microscopy in mature OCs showed a range of intracellular infection that correlated inversely with S. aureus-phagolysosome colocalization. The propensity of OCs to become infected, paired with their diminished bactericidal capacity compared to BMMs, could promote OM progression by allowing S. aureus to evade initial immune regulation and proliferate at the periphery of lesions where OCs are most abundant.IMPORTANCE The inflammation of bone tissue is called osteomyelitis, and most cases are caused by an infection with the bacterium Staphylococcus aureus To date, the bone-building cells, osteoblasts, have been implicated in the progression of these infections, but not much is known about how the bone-resorbing cells, osteoclasts, participate. In this study, we show that S. aureus can infect osteoclasts and proliferate inside these cells, whereas bone-residing macrophages, immune cells related to osteoclasts, destroy the bacteria. These findings elucidate a unique role for osteoclasts to harbor bacteria during infection, providing a possible mechanism by which bacteria could evade destruction by the immune system.
Asunto(s)
Osteoclastos/microbiología , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Diferenciación Celular , Células Cultivadas , Femenino , Macrófagos/metabolismo , Masculino , Ratones , Osteoblastos/microbiología , Osteomielitis/metabolismo , Osteomielitis/microbiología , Fagosomas/metabolismo , Ligando RANK/metabolismo , Staphylococcus aureus/efectos de los fármacosRESUMEN
A prototype line probe assay (LiPA) for identifying hepatitis B virus (HBV) precore variants (INNO-LiPA HBV precore) was evaluated using a panel of 50 sera from 46 patients with HBV infection. The assay detected sequence variations detected commonly in the precore promoter region and in amino acid codons 28 and 29 of the precore gene. There was strong agreement between INNO-LiPA HBV precore results and those of a codon 28 point mutation assay (PMA), with identical results obtained in 40 of 43 sera (93%) typeable by both assays (kappa coefficient (kappa)=0.90). In addition, the precore codon 29 sequence identified by the INNO-LiPA HBV precore was confirmed by nucleotide sequencing in all seven samples analysed. However, the INNO-LiPA HBV precore identified precore promoter sequences much less efficiently. The prototype assay could identify codon 28/29 sequences from as little as 10 HBV genome equivalents in 10 microl serum, and in experiments using artificially prepared mixtures of variants could identify a minor component constituting 2.5% of the total viral DNA population. The INNO-LiPA HBV precore was also straightforward technically and rapid, and is therefore likely to be useful for epidemiological investigations into the prevalence, distribution and clinical significance of HBV precore variants.
Asunto(s)
Variación Genética , Antígenos del Núcleo de la Hepatitis B/sangre , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/virología , Precursores de Proteínas/sangre , Sondas de ADN , ADN Viral/análisis , Antígenos del Núcleo de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Humanos , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Precursores de Proteínas/genética , Juego de Reactivos para Diagnóstico , Tiras Reactivas , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
The long-term outcome of treatment with Interferon Alpha 2B with and without Prednisolone priming in children infected perinatally with hepatitis B was reviewed. The group studied included 48 children (aged 2-16 years), who were HBe antigen and hepatitis B DNA positive between 1991 and 1993. Twenty children were randomized to a therapeutic trial at that time, and received Prednisolone in reducing doses for 6 weeks and Interferon for 16 weeks while 22 children were monitored without treatment for 12 months. Fourteen of the untreated group and 6 additional children later received treatment with Interferon alone (n = 20). Eight children for whom treatment was declined were followed long term. Median follow-up was 7.5 years (range 1.5-10.6). There was no significant effect of Interferon therapy on seroconversion with or without Prednisolone at 12 months post-treatment compared to untreated children. On longer term follow-up, the 5-year HBeAg to anti-HBe seroconversion percentages, estimated from Kaplan-Meier curves, were 54% for Prednisolone plus Interferon, 22% for Interferon alone, and 12% for untreated children. The median time to seroconversion was 3.9 years (range 0.4-8.2) and was shortest in those treated with Prednisolone plus Interferon. Children who had elevated hepatic transaminase enzymes prior to treatment or during Prednisolone priming had a better response. In contrast to many European studies, no child cleared HBsAg and produced anti-HBs. Treatment with Prednisolone priming and Interferon, improved both the time and rate of seroconversion compared to no treatment or Interferon alone, suggesting that this combination of drugs might have an immunomodulatory effect.