RESUMO
Carbapenem-resistant Klebsiella pneumoniae sequence type 258 (CRKP-ST258) can cause chronic infections in lungs and airways, with repeated episodes of bacteremia. In this report we addressed whether the recruitment of myeloid cells producing the anti-inflammatory cytokine interleukin-10 (IL-10) modulates the clearance of CKRP-ST258 in the lungs and establishes bacterial persistence. Our data demonstrate that during pneumonia caused by a clinical isolate of CRKP-ST258 (KP35) there is an early recruitment of monocyte-myeloid-derived suppressor cells (M-MDSCs) and neutrophils that actively produce IL-10. However, M-MDSCs were the cells that sustained the production of IL-10 over the time of infection evaluated. Using mice unable to produce IL-10 (IL-10-/-), we observed that the production of this cytokine during the infection caused by KP35 is important to control bacterial burden, to prevent lung damage, to modulate cytokine production, and to improve host survival. Importantly, intranasal transfer of bone marrow-derived M-MDSCs from mice able to produce IL-10 at 1 day prior to infection improved the ability of IL-10-/- mice to clear KP35 in the lungs, decreasing their mortality. Altogether, our data demonstrate that IL-10 produced by M-MDSCs is required for bacterial clearance, reduction of lung tissue damage, and host survival during KP35 pneumonia.
Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos/imunologia , Interleucina-10/imunologia , Infecções por Klebsiella/imunologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/imunologia , Células Supressoras Mieloides/imunologia , Fatores de Virulência/imunologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Recent reports indicate that astrocytes expressing the mutations of superoxide dismutase-1 (SOD1) may contribute to motor neuron injury in ALS. Here, we provide evidence that mitochondrial dysfunction in SOD1(G93A) rat astrocytes causes astrocytes to induce apoptosis of motor neurons. Mitochondria from SOD1(G93A) rat astrocytes displayed a defective respiratory function, including decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential. Protein 3-nitrotyrosine was detected immunochemically in mitochondrial proteins from SOD1(G93A) astrocytes, suggesting that mitochondrial defects were associated with nitroxidative damage. Furthermore, superoxide radical formation in mitochondria was increased in SOD1(G93A) astrocytes. Similar defects were found in mitochondria isolated from the spinal cord of SOD1(G93A) rats, and pretreatment of animals with the spin trap 5,5-dimethyl-1-pyrroline N-oxide restored mitochondrial function, forming adducts with mitochondrial proteins in vivo. As shown previously, SOD1(G93A) astrocytes induced death of motor neurons in cocultures, compared with nontransgenic ones. This behavior was recapitulated when nontransgenic astrocytes were treated with mitochondrial inhibitors. Remarkably, motor neuron loss was prevented by preincubation of SOD1(G93A) astrocytes with antioxidants and nitric oxide synthase inhibitors. In particular, low concentrations (approximately 10 nm) of two mitochondrial-targeted antioxidants, ubiquinone and carboxy-proxyl nitroxide, each covalently coupled to a triphenylphosphonium cation (Mito-Q and Mito-CP, respectively), prevented mitochondrial dysfunction, reduced superoxide production in SOD1(G93A) astrocytes, and restored motor neuron survival. Together, our results indicate that mitochondrial dysfunction in astrocytes critically influences motor neuron survival and support the potential pharmacological utility of mitochondrial-targeted antioxidants in ALS treatment.
Assuntos
Antioxidantes/administração & dosagem , Astrócitos/enzimologia , Mitocôndrias/enzimologia , Neurônios Motores/enzimologia , Degeneração Neural/enzimologia , Superóxido Dismutase/genética , Substituição de Aminoácidos/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/prevenção & controle , Animais , Animais Geneticamente Modificados , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Células Cultivadas , Sistemas de Liberação de Medicamentos/métodos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Degeneração Neural/genética , Degeneração Neural/prevenção & controle , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/fisiologiaRESUMO
Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Apoptose/fisiologia , Astrócitos/efeitos dos fármacos , Fator 1 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios Motores/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Medula Espinal/citologia , Fatores Etários , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Caspase 3 , Contagem de Células , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Fator 1 de Crescimento de Fibroblastos/metabolismo , Imunofluorescência , Sequestradores de Radicais Livres/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Metaloporfirinas/farmacologia , Camundongos , Camundongos Transgênicos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/imunologia , Fator de Crescimento Neural/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fator de Crescimento Neural/imunologia , Superóxido Dismutase/genética , Tirosina/análogos & derivados , Ureia/análogos & derivadosRESUMO
Reactive astrocytes frequently surround degenerating motor neurons in patients and transgenic animal models of amyotrophic lateral sclerosis (ALS). We report here that reactive astrocytes in the ventral spinal cord of transgenic ALS-mutant G93A superoxide dismutase (SOD) mice expressed nerve growth factor (NGF) in regions where degenerating motor neurons expressed p75 neurotrophin receptor (p75(NTR)) and were immunoreactive for nitrotyrosine. Cultured spinal cord astrocytes incubated with lipopolysaccharide (LPS) or peroxynitrite became reactive and accumulated NGF in the culture medium. Reactive astrocytes caused apoptosis of embryonic rat motor neurons plated on the top of the monolayer. Such motor neuron apoptosis could be prevented when either NGF or p75(NTR) was inhibited with blocking antibodies. In addition, nitric oxide synthase inhibitors were also protective. Exogenous NGF stimulated motor neuron apoptosis only in the presence of a low steady state concentration of nitric oxide. NGF induced apoptosis in motor neurons from p75(NTR +/+) mouse embryos but had no effect in p75(NTR -/-) knockout embryos. Culture media from reactive astrocytes as well as spinal cord lysates from symptomatic G93A SOD mice-stimulated motor neuron apoptosis, but only when incubated with exogenous nitric oxide. This effect was prevented by either NGF or p75(NTR) blocking-antibodies suggesting that it might be mediated by NGF and/or its precursor forms. Our findings show that NGF secreted by reactive astrocytes induce the death of p75-expressing motor neurons by a mechanism involving nitric oxide and peroxynitrite formation. Thus, reactive astrocytes might contribute to the progressive motor neuron degeneration characterizing ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Apoptose , Astrócitos/metabolismo , Neurônios Motores/metabolismo , Fator de Crescimento Neural/biossíntese , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Astrócitos/patologia , Extratos Celulares/farmacologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Fator de Crescimento Neural/antagonistas & inibidores , Fator de Crescimento Neural/metabolismo , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Ratos , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/antagonistas & inibidores , Receptores de Fator de Crescimento Neural/biossíntese , Medula Espinal/química , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase/genéticaRESUMO
The vulnerability of oligodendrocytes to excitatory amino acids may account for the pathology of white matter occurring following hypoxia/ischemia or autoimmune attack. Here, we examined the vulnerability of immature oligodendrocytes (positively labeled by galactocerobroside-C and not expressing myelin basic protein) from neonatal rat spinal cord to kainate, an agonist of excitatory amino acid receptors that induces long-lasting inward currents in immature oligodendrocytes. In particular, we studied whether kainate toxicity was linked to the endogenous production of nitric oxide. We found cultured oligodendrocytes to be highly sensitive to 24-48 h exposure to 0.5-1 mM kainate. The toxin induced striking morphological changes in oligodendrocytes, characterized by the disruption of the process network around the cell body and the growth of one or two long, thick and non-branched processes. A longer exposure to kainate resulted in massive death of oligodendrocytes, which was prevented by 6,7, dinitroquinoxaline-2,3-dione (DNQX) (30 micro M), the antagonist of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic/kainate receptors. Remarkably, we found that those oligodendrocytes displaying bipolar morphology following kainate exposure, also expressed the inducible form of nitric oxide synthase (iNOS) and nitrotyrosine immunoreactivity, suggesting that peroxynitrite could be formed by the reaction of nitric oxide with superoxide. Moreover, kainate toxicity was significantly prevented by addition of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME), further suggesting that nitric oxide-derived oxidants contribute to excitotoxic mechanisms in immature oligodendrocytes.