RESUMEN
Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naïve astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell-astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca2+ increase in astrocytes by promoting glial release of ATP. ATP propagated Ca2+ elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca2+ increase is specifically triggered by the autoreactive CD4+ T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca2+ signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell-astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease.
Asunto(s)
Astrocitos/metabolismo , Señalización del Calcio , Inmunidad Celular , Receptores Purinérgicos/inmunología , Adenosina Trifosfato/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Neuroglía/metabolismo , Ratas , Receptores Purinérgicos P2X7/inmunología , Receptores Purinérgicos P2X7/metabolismo , Transducción de Señal , Médula Espinal/citología , Médula Espinal/inmunologíaRESUMEN
Ethyl pyruvate is a redox analogue of dimethyl fumarate (Tecfidera), a drug for multiple sclerosis treatment. We have recently shown that ethyl pyruvate ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. It affects encephalitogenic T cells and macrophages in vitro, as well as in lymph nodes draining the site of encephalitogenic immunization and within the central nervous system (CNS). Here, in vivo effects of ethyl pyruvate on EAE are thoroughly investigated in the CNS and within the gut associated lymphoid tissue. Ethyl pyruvate reduced infiltrates within the CNS and number of activated macrophages/microglia (ED1+/Iba1+) and proliferating astrocytes (GFAP+). Furthermore, it reduced expression of HMGB1 in activated macrophages/microglia. It also reduced number of activated T cells and antigen-presenting cells and expression of Th1/Th17-related molecules in mesenteric lymph nodes and Peyer's patches. These results contribute to our understanding of anti-encephalitogenic effects of ethyl pyruvate as they provide evidence of its effects within the CNS and imply that these effects are related to reduction of inflammatory immune response in gut associated lymphoid tissue.
Asunto(s)
Sistema Nervioso Central/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Tracto Gastrointestinal/efectos de los fármacos , Piruvatos/uso terapéutico , Animales , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/patología , Proteína HMGB1/antagonistas & inhibidores , Proteína HMGB1/biosíntesis , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ganglios Linfáticos Agregados/efectos de los fármacos , Ganglios Linfáticos Agregados/metabolismo , Ganglios Linfáticos Agregados/patología , Piruvatos/farmacología , Ratas , Resultado del TratamientoRESUMEN
Cell-based tolerogenic therapy is a promising approach for the treatment of autoimmune diseases and transplant rejection. Regulatory T cells and tolerogenic dendritic cells have been particularly explored in the treatment of various autoimmune disorders in experimental models of disease. Although some of these cells have already been tested in a limited number of clinical trials, there is still a need for preclinical research on tolerogenic cells in animal models of autoimmunity. This review will focus on the relevance of data obtained from studies in experimental animal models for the use of tolerogenic cell-based therapy in humans. Also, perspectives for further improvement of tolerogenic cell preparation towards enhanced suppressive activity and stability of the cells will be discussed.
Asunto(s)
Artritis Reumatoide/terapia , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Diabetes Mellitus Tipo 1/terapia , Modelos Animales de Enfermedad , Esclerosis Múltiple/terapia , Animales , Artritis Reumatoide/inmunología , Autoinmunidad/efectos de los fármacos , Autoinmunidad/inmunología , Diabetes Mellitus Tipo 1/inmunología , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/inmunología , Inmunosupresores/administración & dosificación , Esclerosis Múltiple/inmunología , Compuestos Orgánicos/administración & dosificación , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.
RESUMEN
Multiple sclerosis is a chronic inflammatory disease of the central nervous system (CNS). It is widely accepted that autoimmune response against the antigens of the CNS is the essential pathogenic force in the disease. It has recently become increasingly appreciated that activated encephalitogenic cells tend to migrate toward gut associated lymphoid tissues (GALTs) and that interrupted balance between regulatory and inflammatory immunity within the GALT might have decisive role in the initiation and propagation of the CNS autoimmunity. Gut microbiota composition and function has the major impact on the balance in the GALT. Thus, our aim was to perform analyses of gut microbiota in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Albino Oxford (AO) rats that are highly resistant to EAE induction and Dark Agouti (DA) rats that develop EAE after mild immunization were compared for gut microbiota composition in different phases after EAE induction. Microbial analyses of the genus Lactobacillus and related lactic acid bacteria showed higher diversity of Lactobacillus spp. in EAE-resistant AO rats, while some members of Firmicutes and Proteobacteria (Undibacterium oligocarboniphilum) were detected only in feces of DA rats at the peak of the disease (between 13 and 16 days after induction). Interestingly, in contrast to our previous study where Turicibacter sp. was found exclusively in non-immunized AO, but not in DA rats, in this study it was detected in DA rats that remained healthy 16 days after induction, as well as in four of 12 DA rats at the peak of the disease. Similar observation was obtained for the members of Lachnospiraceae. Further, production of a typical regulatory cytokine interleukin-10 was compared in GALT cells of AO and DA rats, and higher production was observed in DA rats. Our data contribute to the idea that gut microbiota and GALT considerably influence multiple sclerosis pathogenesis.
RESUMEN
Cucurbitacin E (CucE) is a highly oxidized steroid consisting of a tetracyclic triterpene. It is a member of a Cucurbitacin family of biomolecules that are predominantly found in Cucurbitaceae plants. CucE has already been identified as a potent anti-inflammatory compound. Here, its effects on CD4(+) T helper (Th) cells and macrophages, as the major encephalitogenic cells in the autoimmunity of the central nervous system, were investigated. Production of major pathogenic Th cell cytokines: interferon-gamma and interleukin-17 were inhibited under the influence of CucE. The effects of CucE on CD4(+) T cells were mediated through the modulation of aryl hydrocarbon receptor, STAT3, NFκB, p38 MAPK, and miR-146 signaling. Further, production of nitric oxide and reactive oxygen species, as well as phagocytic ability, were inhibited in macrophages treated with CucE. These results imply that CucE possesses powerful antiencephalitogenic activity.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Triterpenos/administración & dosificación , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-17/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Transducción de Señal/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Pez CebraRESUMEN
MicroRNAs (miR) are small non-coding RNAs involved in the immune response regulation. miR-155 has been attributed a major pro-inflammatory role in the pathogenesis of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Here, a role of miR-155 in re-activation of encephalitogenic CD4(+) T cells was investigated. Dark Agouti rats were immunized with myelin basic protein (MBP) emulsified in complete Freund's adjuvant. CD4(+) T cells were purified from draining lymph node cells (DLNC) obtained in the inductive phase and from spinal cord immune cells (SCIC) isolated at the peak of EAE. CD4(+) T cells obtained from SCIC (i.e., in vivo re-activated cells) had markedly higher expression of miR-155 in comparison to those purified from DLNC (not re-activated). Likewise, in vitro re-activation of DLNC with MBP led to increase in miR-155 expression. Further, DLNC and DLNC CD4(+) T cells were transfected with an inhibitor of miR-155 during in vitro re-activation. As a result, expression of important CD4(+) T cell effector cytokines IFN-γ and IL-17, but not of regulatory cytokines IL-10 and TGF-ß, was reduced. These results imply that miR-155 supports re-activation of encephalitogenic CD4(+) T cells. Our results contribute to a view that miR-155 might be a valuable target in multiple sclerosis therapy.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Encefalomielitis Autoinmune Experimental/fisiopatología , MicroARNs/genética , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Regulación de la Expresión Génica , Esclerosis Múltiple/genética , Esclerosis Múltiple/fisiopatología , Proteína Básica de Mielina/inmunología , Ratas , Médula Espinal/inmunologíaRESUMEN
Infiltration of macrophages into the central nervous system and activation of microglia are hallmarks of multiple sclerosis and its animal model-experimental autoimmune encephalomyelitis (EAE). Cell death in EAE has been demonstrated as an essential mechanism in the local regulation of the inflammatory reaction, but also as one of the major factors contributing to the destruction of the nervous tissue. The focus of this study was on detection of cell death among ED1(+) cells (macrophages/activated microglia) in the spinal cord of Dark Agouti rats at the peak of EAE. Cell death was assessed using the TUNEL assay and immunostaining for cleaved caspase 3, as markers for cell death in general and "classical" apoptosis, respectively. Major infiltrates of immune cells were detected both in white matter and gray matter of spinal cords in rats at the disease peak. ED1, TUNEL, and caspase 3 positive cells were detected within, but also outside the infiltrates. There were more dying ED1(+) cells in white matter than in gray matter, both in the general population and in infiltrated regions. The observed discrepancy in the proportion of dying ED1(+) cells in spinal cord gray and white matter indicated that in EAE rat macrophages/microglia within gray matter are less prone to cell death induction. This is of special interest in the context of the increasingly appreciated contribution of spinal cord gray matter inflammation to multiple sclerosis pathogenesis. Our findings suggest that activated macrophages/microglia of gray matter are less susceptible to cell death induction. Alternatively, it can be assumed that intrinsic cell death-inductive mechanisms of nervous tissue differ in white and gray matter. Thus, further research on the gray matter macrophages/microglia cell death during EAE is warranted. They should be aimed at identification of the reasons for the observed differences and finding suitable ways to stimulate gray matter activated macrophages/microglia death.
RESUMEN
Covalent attachment of NO to the first approved HIV protease inhibitor Saquinavir (Saq-NO) expands the therapeutic potential of the original drug. Apart from retained antiviral activity, the modified drug exerts strong antitumor effects and lower toxicity. In the present study, we have evaluated the sensitivity of different hematological malignancies to Saq-NO. Saq-NO efficiently diminished the viability of Jurkat, Raji, HL-60 and K562 cells. While Jurkat and Raji cells (established from pediatric patients) displayed abrogated proliferative potential, HL-60 and K652 cells (originated from adults) exposed to Saq-NO treatment underwent caspase dependent apoptosis. In addition, similar sensitivity to Saq-NO was observed in mononuclear blood cells obtained from pediatric patients with acute lymphoblastic leukemia (ALL) and adult patients with acute myeloid leukemia (AML). Western blot analysis indicated p70S6 kinase as a possible intracellular target of Saq-NO action. Moreover, the addition of a NO moiety to Lopinavir resulted in improved antitumor potential as compared to the parental compound, suggesting that NO-derived HIV protease inhibitors are a potential new source of anticancer drugs with unique mode of action.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Quinasas S6 Ribosómicas 70-kDa/efectos de los fármacos , Saquinavir/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Células HL-60 , Humanos , Concentración 50 Inhibidora , Leucemia Mieloide Aguda/enzimología , Óxido Nítrico , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologíaRESUMEN
Ethyl pyruvate (EP) has been increasingly appreciated as an anti-inflammatory and neuroprotective agent with potent pharmacological properties relevant for treatment of various CNS disorders. Microglial cells seem to be particularly sensitive to its effects. In this study, microglial cells were exposed to EP for relatively short periods (10-120min) and inflammatory properties of the cells were determined after 24h of cultivation. Application of EP in the short-term periods inhibited production of interleukin-6, tumor necrosis factor and nitric oxide in microglial cells. At the same time, the effects on cell viability, reactive oxygen species generation and expression of F4/80 and CD40 of microglial cells were minor. NFκB activation was not affected by EP in the cells during the short exposures, thus implying that the observed effect of EP on cytokine and nitric oxide generation was performed in NFκB independent way. Importantly, effects of the short term EP treatment on microglial cells were detected by a real time cell analysis, as well. The observed ability of EP to affect microglial cell function after relatively short time of exposure is relevant for its therapeutic potential against inflammatory disorders of the CNS.
Asunto(s)
Antiinflamatorios/farmacología , Microglía/metabolismo , Piruvatos/farmacología , Animales , Antígenos CD40/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Interleucina-6/biosíntesis , Ratones , Microglía/efectos de los fármacos , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
It has been increasingly appreciated that tumor necrosis factor (TNF) performs various protective and anti-inflammatory functions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Recently, CXCL12 has been identified as a key inhibitor of leukocyte entry into the central nervous system (CNS) and as a regulator of inflammation resulting from the invasion. Here, a positive correlation between expression of TNF and CXCL12 in the CNS samples of EAE rats is presented. Also, it is shown that TNF potentiates CXCL12 expression in astrocytes. These results contribute to a view that TNF produced within the CNS plays a protective role in neuroinflammation.
Asunto(s)
Astrocitos/metabolismo , Quimiocina CXCL12/biosíntesis , Encefalomielitis Autoinmune Experimental/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Modelos Animales de Enfermedad , Inflamación/inmunología , Mediadores de Inflamación/inmunología , Leucocitos/metabolismo , Esclerosis Múltiple/inmunología , Ratas , Médula Espinal/metabolismoRESUMEN
Dimethyl fumarate (DMF), a new drug for multiple sclerosis (MS) treatment, acts against neuroinflammation via mechanisms that are triggered by adduct formation with thiol redox switches. Ethyl pyruvate (EP), an off-the-shelf agent, appears to be a redox analog of DMF, but its immunomodulatory properties have not been put into the context of MS therapy. In this article, we examined and compared the effects of EP and DMF on MS-relevant activity/functions of T cells, macrophages, microglia, and astrocytes. EP efficiently suppressed the release of MS signature cytokines, IFN-γ and IL-17, from human PBMCs. Furthermore, the production of these cytokines was notably decreased in encephalitogenic T cells after in vivo application of EP to rats. Production of two other proinflammatory cytokines, IL-6 and TNF, and NO was suppressed by EP in macrophages and microglia. Reactive oxygen species production in macrophages, microglia activation, and the development of Ag-presenting phenotype in microglia and macrophages were constrained by EP. The release of IL-6 was reduced in astrocytes. Finally, EP inhibited the activation of transcription factor NF-κB in microglia and astrocytes. Most of these effects were also found for DMF, implying that EP and DMF share common targets and mechanisms of action. Importantly, EP had in vivo impact on experimental autoimmune encephalomyelitis, an animal model of MS. Treatment with EP resulted in delay and shortening of the first relapse, and lower clinical scores, whereas the second attack was annihilated. Further studies on the possibility to use EP as an MS therapeutic are warranted.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Fumaratos/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Piruvatos/farmacología , Animales , Antiinflamatorios/farmacología , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Dimetilfumarato , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Humanos , Immunoblotting , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismo , FN-kappa B/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Covalent attachment of the nitric oxide (NO) moiety to the HIV protease inhibitor Saquinavir (Saq) produced a new chemical entity, named Saquinavir-NO, (Saq-NO) with reduced toxicity and potent immunoregulatory influence on T lymphocytes. In this study, we have compared head-to-head the effects of Saq-NO and Saq on mouse and rat peritoneal macrophage cytokine secretion and NO production upon in vitro, ex vivo and in vivo conditions. The results demonstrate that Saq-NO, but not Saq, potently decreased interleukin (IL)-10, IL-6 and nitrite accumulation and increased the levels of IL-1ß and tumour necrosis factor (TNF) in supernatants of mouse and rat macrophage cultures in vitro. Treatment of mice with Saq-NO, but not Saq, inhibited ex vivo secretion of IL-6 from macrophages. Consistent with these findings, Saq-NO also reduced blood levels of IL-6 in lipopolysaccharide-treated mice. The observed inhibitory influence of Saq-NO on IL-6 generation in macrophages may be involved in the observed antitumour and immunomodulatory effects of the drug.
Asunto(s)
Interleucina-6/biosíntesis , Macrófagos Peritoneales/efectos de los fármacos , Saquinavir/análogos & derivados , Animales , Células Cultivadas , Interleucina-10/antagonistas & inhibidores , Interleucina-10/biosíntesis , Interleucina-1beta/biosíntesis , Interleucina-6/antagonistas & inhibidores , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , Ratas , Saquinavir/farmacología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Iridium(III) complexes of the type [Ir(η(5) -C5 Me5 )Cl2 {Ph2 PCH2 CH2 CH2 S(O)x Ph-κP}] (x=0-2; 1-3) and [Ir(η(5) -C5 Me5 )Cl{Ph2 PCH2 CH2 CH2 S(O)x Ph-κP,κS}][PF6 ] (x=0-1; 4 and 5) with 3-(diphenylphosphino)propyl phenyl sulfide, sulfoxide, and sulfone ligands Ph2 PCH2 CH2 CH2 S(O)x Ph were designed, synthesized, and characterized fully, including X-ray diffraction analyses for complexes 3 and 4. In vitro studies against human thyroid carcinoma (8505C), submandibular carcinoma (A253), breast adenocarcinoma (MCF-7), colon adenocarcinoma (SW480), and melanoma (518A2) cell lines provided evidence for the high biological potential of the neutral and cationic iridium(III) complexes. Neutral iridium(III) complex 5 proved to be the most active, with IC50 values up to about 0.1â µM, representing activities of up to one order of magnitude higher than cisplatin. Using 8505C cells, apoptosis was shown to be the main mechanism through which complex 5 exerts its tumoricidal action. The described iridium(III) complexes represent potential leads in the search for novel metal-based anticancer agents.
Asunto(s)
Antineoplásicos/química , Complejos de Coordinación/química , Iridio/química , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/farmacología , Cristalografía por Rayos X , Humanos , Ligandos , Células MCF-7 , Conformación Molecular , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-ActividadRESUMEN
Neutral iridium(III) complexes of the type [Ir(η(5)-C5Me5)Cl2{Ph2PCH2S(O)xPh-κP}] (1-3) with diphenylphosphino-functionalized methyl phenyl sulfides, sulfoxides, and sulfones Ph2PCH2S(O)xPh (x = 0, L1; 1, L2; 2, L3) and the cationic complex [Ir(η(5)-C5Me5)Cl{Ph2PCH2SPh-κP,κS}][PF6] (4) were synthesized and fully characterized analytically and spectroscopically. Furthermore, the structure of 2 was determined by X-ray diffraction analysis. The biological potential of the neutral and cationic iridium(III) complexes was tested in vitro against the cell lines 8505C, A253, MCF-7, SW480 and 518A2. Complex [Ir(η(5)-C5Me5)Cl2{Ph2PCH2S(O)Ph-κP}] (2), with ligand L2 κP coordinated containing a pendent sulfinyl group, is the most active one (IC50 values of about 3 µM), thus, with activities comparable to cisplatin. Complex 2 proved to have an even a higher antiproliferative activity than cisplatin against 8505C and SW480 cell lines, used as a model system of highly anaplastic cancers with low sensitivity to conventional chemotherapeutics such as cisplatin. Additional experiments demonstrated that apoptosis and autophagic cell death contribute to the drug's tumoricidal action.
Asunto(s)
Antineoplásicos/farmacología , Iridio/química , Compuestos Organometálicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Cationes/síntesis química , Cationes/química , Cationes/farmacología , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/química , Cisplatino/farmacología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ligandos , Células MCF-7 , Modelos Moleculares , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-ActividadRESUMEN
Rat models of pulmonary aspergillosis are used widely in diagnostic studies and in exploring antifungal therapeutic modalities, but there is lack of data concerning antifungal immunity in rats. In this study, cytokine response to pulmonary infection to Aspergillus fumigatus in non-immunosuppressed rats is explored. Temporal display (from the start of infection up to its eradication) of proinflammatory cytokines (IFN-γ and IL-17) as well as Th2/anti-inflammatory ones (IL-4 and IL-10) was explored by measuring their presence in the environment in which elimination of infection occur (lung homogenates), by production of these mediators by lung cells (recovered by enzyme digestion or by bronchoalveolar lavage) as well as by cells of draining lymph nodes (as sites of generation of cytokine-producing cells). Reduction of infection (1 × 107conidia) was associated with an increase of IFN-γ and IL-17 content in lung homogenates, but with unchanged IL-4 and IL-10 content. Lung cells produced proinflammatory cytokines with differential dynamics (IFN-γ earlier than IL-17). Differential pattern of Th2/anti-inflammatory cytokine production by lung cells was observed (unchanged IL-4 and increased IL-10), with the levels of the latter higher than proinflammatory cytokines. Upregulation of IFN-γ, IL-17 and IL-10 production and gene expression, but downregulation of IL-4, by draining lymph node cells (dLN cells) accounted essentially for the observed ex vivo cytokine response in lungs. Similar pattern of cytokine production by dLN cells following restimulation with A. fumigatus conidia confirmed the specificity of cytokine response to the fungus. Draining lymph node CD4⺠cells seems to be the main source of proinflammatory cytokines, significant contributors to IL-10 production and the target for down regulation of IL-4. The knowledge of immune-based mechanisms of defense against A. fumigatus in rats might be helpful in the future use of rat models of pulmonary aspergillosis particularly those that develop immune-based therapeutic interventions as an adjunct treatment of fungal diseases.
Asunto(s)
Aspergillus/inmunología , Linfocitos T CD4-Positivos/inmunología , Pulmón/inmunología , Ganglios Linfáticos/inmunología , Aspergilosis Pulmonar/inmunología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Inmunocompetencia , Inmunomodulación , Masculino , Ratas , Balance Th1 - Th2RESUMEN
Transferrin (Tf) has a major role in T cell activation and proliferation. Here, we investigated whether Tf exerts immunomodulatory effects on T cells and in development of T-cell driven experimental autoimmune encephalomyelitis (EAE). While treatment of concanavalin A-stimulated splenocytes with apotransferrin (ApoTf) did not affect release of IL-1ß, TNF, IFN-γ, IL-17, IL-4, and IL-10, it markedly and dose-dependently down-regulated synthesis of IL-2 in these cells. ApoTf also inhibited IL-2 generation in purified CD3+ T cells and the effect was accompanied with down-regulation of MAPK p44/42 and NFκB signaling. Despite impeded IL-2 release, proliferation of splenocytes was not inhibited by ApoTf. Importantly, ApoTf ameliorated EAE in mice and significantly reduced ex vivo IL-2 production in proteolipid protein-specific lymphocytes. Thus ApoTf may be a promising beneficial agent for multiple sclerosis.
Asunto(s)
Apoproteínas/fisiología , Regulación hacia Abajo/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/prevención & control , Interleucina-2/antagonistas & inhibidores , Interleucina-2/biosíntesis , Transferrina/fisiología , Animales , Apoproteínas/administración & dosificación , Células Cultivadas , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Inmunosupresores/administración & dosificación , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos NOD , FN-kappa B/antagonistas & inhibidores , Transducción de Señal/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transferrina/administración & dosificaciónRESUMEN
Chemokine CXCL12 (C-X-C motif chemokine ligand 12) restricts immune cell invasion of the central nervous system (CNS) and limits neuroinflammation in experimental autoimmune encephalomyelitis (EAE), an animal model of inflammatory and demyelinating disease of the CNS, multiple sclerosis (MS). Nitric oxide (NO), by contrast, predominantly contributes to CNS tissue destruction in MS and EAE. Thus, the influence of NO on CXCL12 in the inflamed CNS was investigated. Excess expression of inducible NO synthase was inversely correlated to CXCL12 gene expression in spinal cord homogenates of rats immunized to develop EAE. NO inhibited gene expression of CXCL12 in astrocytes and endothelial cells in vitro. The inhibition was paralleled with reduction of p38 mitogen-activated protein kinase (MAPK) phosphorylation and it was mimicked with inhibitors of p38 MAPK activation in astrocytes. In vivo suppression of nitric generation recovered CXCL12 expression in the CNS and attenuated EAE in Dark Agouti rats. On the contrary, in vivo NO donation decreased CXCL12 expression in the CNS of EAE-resistant Albino Oxford (AO) rats. However, the effect was not paralleled with induction of EAE in AO rats. It is suggested that NO acting through suppression of p38 MAPK inhibits CXCL12 expression in neuroinflammation. These results imply that downregulation of NO release and protection of CXCL12 expression within the CNS might present the potential approaches in MS therapy.
Asunto(s)
Quimiocina CXCL12/antagonistas & inhibidores , Encefalomielitis Autoinmune Experimental/inmunología , Células Endoteliales/inmunología , Esclerosis Múltiple/inmunología , Óxido Nítrico/inmunología , Animales , Astrocitos/inmunología , Quimiocina CXCL12/biosíntesis , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/terapia , Humanos , Inmunoterapia/métodos , Inmunoterapia/tendencias , Esclerosis Múltiple/terapia , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas , Ratas Endogámicas , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Dark Agouti (DA) rats are highly susceptible to induction of experimental autoimmune encephalomyelitis (EAE), still they completely recover from the disease. Here, we were interested to determine contribution of major anti-inflammatory cytokines transforming growth factor (TGF)-ß and interleukin (IL)-10 to the recovery of DA rats from EAE. To that extent we determined CNS expression of these cytokines in DA rats at different phases of EAE and compared data to those obtained in EAE-resistant Albino Oxford (AO) rats. Higher expression of TGF-ß was persistently observed in the CNS of AO rats, even if rats were not immunized. This implied that high TGF-ß within the CNS is important for resistance of AO rats to EAE induction. On the contrary, IL-10 expression was consistently higher in DA than in AO rats and it culminated at the peak of EAE. Methylprednisolone suppressed EAE and expression of IL-10 in spinal cord homogenates, while IL-10 was increased in CNS-infiltrating immune cells. This implied that IL-10 might have a significant role in recovery of DA rats from the disease. Thus, we next explored effects of IL-10 on astrocytes, glial cells that largely contribute to control of CNS inflammation. IL-10 stimulated astrocytic expression of an important regulator of neuroinflammation, CXCL12. Thus, IL-10 might contribute to recovery of DA rats from EAE through induction of CXCL12 expression in astrocytes.
Asunto(s)
Astrocitos/inmunología , Convalecencia , Encefalomielitis Autoinmune Experimental/inmunología , Interleucina-10/metabolismo , Médula Espinal/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Progresión de la Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Interleucina-10/genética , Metilprednisolona/administración & dosificación , Ratas , Ratas Endogámicas , Factor de Crecimiento Transformador beta/genéticaRESUMEN
CXCL12 plays a protective role in CNS autoimmunity. Expression of CXCL12-γ, which has distinct structural and functional properties than the other isoforms of CXCL12, was determined in spinal cords of rats immunized to develop experimental autoimmune encephalomyelitis (EAE). CNS expression of CXCL12-γ was markedly lower in EAE-prone Dark Agouti rats than in EAE-resistant Albino Oxford rats, both in spinal cord homogenates and micro-blood vessels isolated from spinal cords. Inhibition of nitric oxide (NO) synthesis in DA rats upregulated, while donation of NO in AO rats downregulated CNS expression of CXCL12-γ. NO inhibited CXCL12-γ expression in astrocytes in vitro. A splice variant of CXCL12-γ which migrates into nucleolus was not detected in spinal cord or astrocytes. Thus, CXCL12-γ is expressed in the CNS after EAE induction, but its expression is markedly suppressed in spinal cord affected with full blown inflammation. NO is an important regulator of CXCL12-γ expression in neuroinflammation.