RESUMEN
While new treatments have been developed to control joint disease in rheumatoid arthritis, they are partially effective and do not promote structural repair of cartilage. Following an initial identification of α-1-Antitrypsin (AAT) during the resolution phase of acute inflammation, we report here the properties of this protein in the context of cartilage protection, joint inflammation, and associated pain behavior. Intra-articular and systemic administration of AAT reversed joint inflammation, nociception, and cartilage degradation in the KBxN serum and neutrophil elastase models of arthritis. Ex vivo analyses of arthritic joints revealed that AAT promoted transcription of col2a1, acan, and sox9 and downregulated mmp13 and adamts5 gene expression. In vitro studies using human chondrocytes revealed that SERPINA1 transfection and rAAT protein promoted chondrogenic differentiation through activation of PKA-dependent CREB signaling and inhibition of Wnt/ß-catenin pathways. Thus, AAT is endowed with anti-inflammatory, analgesic, and chondroprotective properties that are partially inter-related. We propose that AAT could be developed for new therapeutic strategies to reduce arthritic pain and repair damaged cartilage.
Asunto(s)
Artritis Experimental/complicaciones , Condrocitos/citología , Condrogénesis , Inflamación/prevención & control , Dolor/prevención & control , alfa 1-Antitripsina/farmacología , Animales , Condrocitos/efectos de los fármacos , Inflamación/etiología , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Dolor/etiología , Dolor/patología , Ratas , Ratas WistarRESUMEN
RATIONALE: Specialized pro-resolving mediators (SPM-lipoxins, resolvins, protectins, and maresins) are produced via the enzymatic conversion of essential fatty acids, including the omega-3 fatty acids docosahexaenoic acid and n-3 docosapentaenoic acid. These mediators exert potent leukocyte directed actions and control vascular inflammation. Supplementation of animals and humans with essential fatty acids, in particular omega-3 fatty acids, exerts protective actions reducing vascular and systemic inflammation. Of note, the mechanism(s) activated by these supplements in exerting their protective actions remain poorly understood. OBJECTIVE: Given that essential fatty acids are precursors in the biosynthesises of SPM, the aim of the present study was to establish the relationship between supplementation and peripheral SPM concentrations. We also investigated the relationship between changes in plasma SPM concentrations and peripheral blood platelet and leukocyte responses. METHODS AND RESULTS: Healthy volunteers were enrolled in a double-blinded, placebo-controlled, crossover study, and peripheral blood was collected at baseline, 2, 4, 6, and 24 hours post administration of placebo or one of 3 doses of an enriched marine oil supplement. Assessment of plasma SPM concentrations using lipid mediator profiling demonstrated a time- and dose-dependent increase in peripheral blood SPM concentration. Supplementation also led to a regulation of peripheral blood cell responses. Here we found a dose-dependent increase in neutrophil and monocyte phagocytosis of bacteria and a decrease in the diurnal activation of leukocytes and platelets, as measured by a reduction in adhesion molecule expression. In addition, transcriptomic analysis of peripheral blood cells demonstrated a marked change in transcript levels of immune and metabolic genes 24 hours post supplementation when compared with placebo. CONCLUSIONS: Together, these findings demonstrate that supplementation with an enriched marine oil leads to an increase in peripheral blood SPM concentrations and reprograms peripheral blood cells, indicating a role for SPM in mediating the immune-directed actions of this supplement. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT03347006.
Asunto(s)
Suplementos Dietéticos , Ácidos Docosahexaenoicos/sangre , Ácidos Grasos Omega-3/farmacología , Aceites de Pescado/farmacología , Sistema Inmunológico/efectos de los fármacos , Lipoxinas/sangre , Adulto , Biomarcadores , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Moléculas de Adhesión Celular/sangre , Ritmo Circadiano/efectos de los fármacos , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Ácidos Grasos Esenciales/fisiología , Ácidos Grasos Omega-3/administración & dosificación , Femenino , Aceites de Pescado/administración & dosificación , Ontología de Genes , Humanos , Masculino , Persona de Mediana Edad , Fagocitosis/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Adulto JovenRESUMEN
RATIONALE: Diurnal mechanisms are central to regulating host responses. Recent studies uncovered a novel family of mediators termed as specialized proresolving mediators that terminate inflammation without interfering with the immune response. OBJECTIVE: Herein, we investigated the diurnal regulation of specialized proresolving mediators in humans and their role in controlling peripheral blood leukocyte and platelet activation. METHODS AND RESULTS: Using lipid mediator profiling and healthy volunteers, we found that plasma concentrations of n-3 docosapentaenoic acid-derived D-series resolvins (RvDn-3 DPA) were regulated in a diurnal manner. The production and regulation of these mediators was markedly altered in patients at risk of myocardial infarct. These changes were associated with decreased 5-lipoxygenase expression and activity, as well as increased systemic adenosine concentrations. We also found a significant negative correlation between plasma RvDn-3 DPA and markers of platelet, monocyte, and neutrophil activation, including CD63 and CD11b. Incubation of RvDn-3 DPA with peripheral blood from healthy volunteers and patients with cardiovascular disease significantly and dose-dependently decreased platelet and leukocyte activation. Furthermore, administration of RvD5n-3 DPA to ApoE-/- (apolipoprotein E deficient) mice significantly reduced platelet-leukocyte aggregates, vascular thromboxane B2 concentrations, and aortic lesions. CONCLUSIONS: These results demonstrate that peripheral blood RvDn-3 DPA are diurnally regulated in humans, and dysregulation in the production of these mediators may lead to cardiovascular disease.
Asunto(s)
Ritmo Circadiano , Ácidos Grasos Insaturados/sangre , Infarto del Miocardio/sangre , Adenosina/sangre , Animales , Plaquetas/metabolismo , Humanos , Inflamación/sangre , Leucocitos/metabolismo , Lipooxigenasa/sangre , Ratones , Tromboxano B2/sangreRESUMEN
Rheumatoid arthritis is an inflammatory condition characterized by overzealous inflammation that leads to joint damage and is associated with an increased incidence of cardiovascular disease. Statins are frontline therapeutics for patients with cardiovascular disease and exert beneficial actions in rheumatoid arthritis. The mechanism that mediates the beneficial actions of statins in rheumatoid arthritis remains of interest. In the present study, we found that the administration of 2 clinically relevant statins-atorvastatin (0.2 mg/kg) or pravastatin (0.2 mg/kg)-to mice during inflammatory arthritis up-regulated systemic and tissue amounts of a novel family of proresolving mediators, termed 13-series resolvins (RvTs), and significantly reduced joint disease. Of note, administration of simvastatin (0.2 mg/kg) did not significantly up-regulate RvTs or reduce joint inflammation. We also found that atorvastatin and pravastatin each reduced systemic leukocyte activation, including platelet-monocyte aggregates (â¼25-60%). These statins decreased neutrophil trafficking to the joint as well as joint monocyte and macrophage numbers. Atorvastatin and pravastatin produced significant reductions (â¼30-50%) in expression of CD11b and major histocompatibility complex class II on both monocytes and monocyte-derived macrophages in joints. Administration of an inhibitor to cyclooxygenase-2, the initiating enzyme in the RvT pathway, reversed the protective actions of these statins on both joint and systemic inflammation. Together, these findings provide evidence for the role of RvTs in mediating the protective actions of atorvastatin and pravastatin in reducing local and vascular inflammation, and suggest that RvTs may be useful in measuring the anti-inflammatory actions of statins.-Walker, M. E., Souza, P. R., Colas, R. A., Dalli, J. 13-Series resolvins mediate the leukocyte-platelet actions of atorvastatin and pravastatin in inflammatory arthritis.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Artritis/tratamiento farmacológico , Atorvastatina/uso terapéutico , Ácidos Docosahexaenoicos/metabolismo , Inflamación/tratamiento farmacológico , Pravastatina/uso terapéutico , Animales , Artritis/inducido químicamente , Artritis/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The clinical benefits of short-term therapy with glucocorticoids (GC) in patients with inflammatory bowel disease (IBD) are widely known. However, the effects of this treatment towards the re-establishment of the regulatory network in IBD are not fully explored. We have evaluated the immunological effects of the abbreviated GC therapy in experimental colitis induced by 3% dextran sulphate sodium in C57BL/6 mice. Treatment with GC improved disease outcome, constrained circulating leucocytes and ameliorated intestinal inflammation. The control of the local inflammatory responses involved a reduction in the expression of interferon-γ and interleukin-1ß, associated with augmented mRNA levels of peroxisome proliferator-activated receptors (α and γ) in intestine. Furthermore, there was a reduction of CD4+ T cells producing interferon-γ, together with an increased frequency of the putative regulatory population of T cells producing interleukin-10, in spleen. These systemic alterations were accompanied by a decrease in the proliferative potential of splenocytes of mice treated in vivo with GC. Notably, treatment with GC also led to an increase in the frequency of the regulatory markers GITR, CTLA-4, PD-1, CD73 and FoxP3, more prominently in spleen. Taken together, our results pointed to a role of GC in the control of leucocyte responsiveness and re-establishment of a regulatory system, which probably contributed to disease control and the restoration of immune balance. Finally, this is the first time that GC treatment was associated with the modulation of a broad number of regulatory markers in an experimental model of colitis.
Asunto(s)
Colitis/tratamiento farmacológico , Glucocorticoides/uso terapéutico , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD4/metabolismo , Células Cultivadas , Protocolos Clínicos , Colitis/inducido químicamente , Sulfato de Dextran , Proteína Relacionada con TNFR Inducida por Glucocorticoide/metabolismo , Humanos , Inmunomodulación , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Masculino , Ratones , Receptores Activados del Proliferador del Peroxisoma/metabolismoRESUMEN
OBJECTIVES: Although TB immunotherapy improves the results of conventional drug treatment, the effects of combining chemotherapy and immunotherapy have never been systematically evaluated. We used a comprehensive lung transcriptome analysis to directly compare the activity of combined chemotherapy and immunotherapy with that of single treatments in a mouse model of TB. METHODS: Mycobacterium tuberculosis-infected mice in the chronic phase of the disease (day 30) received: (i) isoniazid and rifampicin (drugs) daily for 30 days; (ii) DNA immunotherapy (DNA), consisting of four 100 µg injections at 10 day intervals; (iii) both therapies (DNAâ+âdrugs); or (iv) saline. The effects were evaluated 10 days after the end of treatment (day 70 post-infection). RESULTS: In all groups a systemic reduction in the load of bacilli was observed, bacilli became undetectable in the drugs and DNAâ+âdrugs groups, but the whole lung transcriptome analysis showed 867 genes exclusively modulated by the DNAâ+âdrugs combination. Gene enrichment analysis indicated that DNAâ+âdrugs treatment provided synergistic effects, including the down-regulation of proinflammatory cytokines and mediators of fibrosis, as confirmed by real-time PCR, ELISA, histopathology and hydroxyproline assay. CONCLUSIONS: Our results provide a molecular basis for the advantages of TB treatment using combined chemotherapy and DNA immunotherapy and demonstrate the synergistic effects obtained with this strategy.
Asunto(s)
Terapia Combinada/métodos , Quimioterapia/métodos , Inmunoterapia/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/inmunología , Tuberculosis/terapia , Animales , Antituberculosos/administración & dosificación , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Isoniazida/administración & dosificación , Pulmón/microbiología , Pulmón/patología , Ratones Endogámicos BALB C , Rifampin/administración & dosificación , Resultado del Tratamiento , Vacunas de ADN/administración & dosificaciónRESUMEN
BACKGROUND: Rheumatoid arthritis (RA) is a progressive degenerative disorder that leads to joint destruction. Available treatments only target the inflammatory component with minimal impact on joint repair. We recently uncovered a previously unappreciated family of pro-resolving mediators, the maresin conjugate in tissue regeneration (MCTR), that display both immunoregulatory and tissue-protective activities. Thus, we queried whether the production of these autacoids is disrupted in RA patients and whether they can be useful in treating joint inflammation and promoting joint repair. METHODS: Using a highly phenotyped RA cohort we evaluated plasma MCTR concentrations and correlated these to clinical markers of disease activity. To evaluate the immunoregulatory and tissue reparative activities we employed both in vivo models of arthritis and organ culture models. FINDINGS: Herein, we observed that plasma MCTR3 concentrations were negatively correlated with joint disease activity and severity in RA patients. Evaluation of the mechanisms engaged by this mediator in arthritic mice demonstrated that MCTR3 reprograms monocytes to confer enduring joint protective properties. Single cell transcriptomic profiling and flow cytometric evaluation of macrophages from mice treated with MCTR3-reprogrammed monocytes revealed a role for Arginase-1 (Arg-1) in mediating their joint reparative and pro-resolving activities. Arg-1 inhibition reversed both the anti-arthritic and tissue reparative actions of MCTR3-reprogrammed monocytes. INTERPRETATION: Our findings demonstrate that circulating MCTR3 levels are negatively correlated with disease in RA. When administered to mice in vivo, MCTR3 displayed both anti-inflammatory and joint reparative activities, protecting both cartilage and bone in murine arthritis. These activities were, at least in part, mediated via the reprogramming of mononuclear phagocyte responses. FUNDING: This work was supported by funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant no: 677542) and the Barts Charity (grant no: MGU0343) to J.D. J.D. is also supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant 107613/Z/15/Z).
Asunto(s)
Artritis Experimental , Artritis Reumatoide , Animales , Antiinflamatorios/farmacología , Arginasa/genética , Artritis Reumatoide/tratamiento farmacológico , Humanos , Macrófagos , Ratones , MonocitosRESUMEN
The plasma level of the chemokine CCL3 is elevated in patients with chronic severe schistosomiasis mansoni. We have previously shown that CCL3(-/-) mice with experimental infection showed diminished pathology and worm burden compared to those of wild-type (WT) mice. To elucidate further the role of CC chemokines during schistosomiasis mansoni infection, we evaluated the course of infection in C57BL/6J mice deficient in CCR5, one of the receptors for CCL3. The CCR5 deficiency proved to be remarkably deleterious to the host, since mortality rates reached 70% at 14 weeks postinfection in CCR5(-/-) mice and 19% in WT mice. The increased lethality was not associated with an increased parasite burden, since similar numbers of eggs and adult worms were found in mice from both groups. Liver granulomas of chronically infected CCR5(-/-) mice were larger and showed greater numbers of cells and collagen deposition than liver granulomas from WT mice. This was associated with higher levels of production of intereleukin-5 (IL-5), IL-13, CCL3, and CCL5 in infected CCR5(-/-) mice than in infected WT mice. Moreover, at 8 weeks after infection, just before changes in pathology and mortality, the numbers of FoxP3-positive cells were lower in liver granulomas of CCR5(-/-) mice than in WT mice. In conclusion, the CCR5 deletion is deleterious to mice infected with Schistosoma mansoni, and this is associated with enhanced fibrosis and granulomatous inflammation.
Asunto(s)
Granuloma/patología , Inflamación/patología , Receptores CCR5/deficiencia , Animales , Modelos Animales de Enfermedad , Femenino , Granuloma/inmunología , Inmunohistoquímica , Inflamación/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores CCR5/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis/inmunología , Esquistosomiasis/patologíaRESUMEN
G-protein-coupled receptor 40 (GPR40) is known to play a role in the regulation of fatty acids, insulin secretion, and inflammation. However, the function of this receptor in human neutrophils, one of the first leukocytes to arrive at the site of infection, remains to be fully elucidated. In the present study, we demonstrate that GPR40 is upregulated on activated human neutrophils and investigated the functional effects upon treatment with a selective agonist; GW9508. Interestingly, GPR40 expression was up-regulated after neutrophil stimulation with platelet-activating factor (10 nM) or leukotriene B4 (LTB4, 10 nM) suggesting potential regulatory roles for this receptor during inflammation. Indeed, GW9508 (1 and 10 µM) increased neutrophil chemotaxis in response to the chemokine IL-8 (30 ng/ml) and enhanced phagocytosis of Escherichia coli by approximately 50% when tested at 0.1 and 1 µM. These results were translated in vivo whereby administration of GW9508 (10 mg/kg, i.p.) during E. coli infections resulted in elevated peritoneal leukocyte infiltration with a higher phagocytic capacity. Importantly, GW9508 administration also modulated the lipid mediator profile, with increased levels of the pro-resolving mediators resolvin D3 and lipoxins. In conclusion, GPR40 is expressed by activated neutrophils and plays an important host protective role to aid clearance of bacterial infections.
Asunto(s)
Infecciones por Escherichia coli/tratamiento farmacológico , Escherichia coli/patogenicidad , Metilaminas/farmacología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Peritonitis/tratamiento farmacológico , Fagocitosis/efectos de los fármacos , Propionatos/farmacología , Receptores Acoplados a Proteínas G/agonistas , Animales , Células Cultivadas , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Ácidos Grasos Insaturados/metabolismo , Interacciones Huésped-Patógeno , Humanos , Lipoxinas/metabolismo , Masculino , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/microbiología , Peritonitis/inmunología , Peritonitis/metabolismo , Peritonitis/microbiología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
Biomarkers are needed for predicting the effectiveness of disease modifying antirheumatic drugs (DMARDs). Here, using functional lipid mediator profiling and deeply phenotyped patients with early rheumatoid arthritis (RA), we observe that peripheral blood specialized pro-resolving mediator (SPM) concentrations are linked with both DMARD responsiveness and disease pathotype. Machine learning analysis demonstrates that baseline plasma concentrations of resolvin D4, 10S, 17S-dihydroxy-docosapentaenoic acid, 15R-Lipoxin (LX)A4 and n-3 docosapentaenoic-derived Maresin 1 are predictive of DMARD responsiveness at 6 months. Assessment of circulating SPM concentrations 6-months after treatment initiation establishes that differences between responders and non-responders are maintained, with a decrease in SPM concentrations in patients resistant to DMARD therapy. These findings elucidate the potential utility of plasma SPM concentrations as biomarkers of DMARD responsiveness in RA.
Asunto(s)
Antirreumáticos/administración & dosificación , Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Líquido Sinovial/efectos de los fármacos , Antirreumáticos/sangre , Artritis Reumatoide/patología , Estudios de Cohortes , Ácidos Docosahexaenoicos/sangre , Ácidos Grasos Insaturados/sangre , Humanos , Lipoxinas/sangre , Resultado del TratamientoRESUMEN
BACKGROUND: The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally. RESULTS: We developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 microg of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-gamma and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 microg). CONCLUSION: Our objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.
Asunto(s)
Proteínas Bacterianas/administración & dosificación , Chaperoninas/administración & dosificación , Mycobacterium tuberculosis , Vacunas contra la Tuberculosis/administración & dosificación , Tuberculosis Pulmonar/inmunología , Vacunas de ADN/administración & dosificación , Administración Intranasal , Animales , Proteínas Bacterianas/inmunología , Chaperonina 60 , Chaperoninas/inmunología , Femenino , Inmunidad Activa/efectos de los fármacos , Inmunización Secundaria , Liposomas , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Células TH1/efectos de los fármacos , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/prevención & controlRESUMEN
Macrophages are central in orchestrating the clearance of apoptotic cells and cellular debris during inflammation, with the mechanism(s) regulating this process remaining of interest. Herein, we found that the n-3 docosapentaenoic acid-derived protectin (PDn-3 DPA) biosynthetic pathway regulated the differentiation of human monocytes, altering macrophage phenotype, efferocytosis, and bacterial phagocytosis. Using lipid mediator profiling, human primary cells and recombinant enzymes we found that human 15-lipoxygenases initiate the PDn-3 DPA pathway catalyzing the formation of an allylic epoxide. The complete stereochemistry of this epoxide was determined using stereocontrolled total organic synthesis as 16S,17S-epoxy-7Z,10Z,12E,14E,19Z-docosapentaenoic acid (16S,17S-ePDn-3 DPA). This intermediate was enzymatically converted by epoxide hydrolases to PD1n-3 DPA and PD2n-3 DPA, with epoxide hydrolase 2 converting 16S,17S-ePDn-3 DPA to PD2n-3 DPA in human monocytes. Taken together these results establish the PDn-3 DPA biosynthetic pathway in human monocytes and macrophages and its role in regulating macrophage resolution responses.
Asunto(s)
Antígenos CD59/metabolismo , Diferenciación Celular , Ácidos Grasos Insaturados/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Macrófagos/fisiología , Araquidonato 15-Lipooxigenasa/metabolismo , Antígenos CD59/antagonistas & inhibidores , Antígenos CD59/química , Diferenciación Celular/efectos de los fármacos , Ácidos Grasos Insaturados/antagonistas & inhibidores , Ácidos Grasos Insaturados/química , Voluntarios Sanos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Inhibidores de la Lipooxigenasa/química , Inhibidores de la Lipooxigenasa/farmacología , Macrófagos/efectos de los fármacos , Estructura Molecular , EstereoisomerismoRESUMEN
The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.
RESUMEN
Galectin-3 has been associated with a plethora of proinflammatory functions because of its ability, among others, to promote neutrophil activation and because of the reduction in neutrophil recruitment in models of infection in Gal-3-null mice. Conversely, it has also been linked to resolution of inflammation through its actions as an opsonin and its ability to promote efferocytosis of apoptotic neutrophils. Using a self-resolving model of peritonitis, we have addressed the modulation and role of Gal-3 in acute inflammation. We have shown that Gal-3 expression is increased in neutrophils that travel to the inflamed peritoneum and that cellular localization of this lectin is modulated during the course of the inflammatory response. Furthermore, neutrophil recruitment to the inflamed peritoneum is increased in Gal-3-null mice during the course of the response, and that correlates with reduced numbers of monocytes/macrophages in the cavities of those mice, as well as reduced apoptosis and efferocytosis of Gal-3-null neutrophils. These data indicate a role for endogenous Gal-3 in neutrophil clearance during acute inflammation.
Asunto(s)
Galectina 3/deficiencia , Inflamación/patología , Neutrófilos/metabolismo , Enfermedad Aguda , Animales , Apoptosis , Membrana Celular/metabolismo , Citosol/metabolismo , Modelos Animales de Enfermedad , Galectina 3/metabolismo , Inflamación/metabolismo , Masculino , Ratones Endogámicos C57BL , Peritonitis/patología , Zimosan/administración & dosificaciónRESUMEN
BACKGROUND: CD163, receptor for the haptoglobin-hemoglobin complex, is expressed on monocytes/macrophages and neutrophils. A soluble form of CD163 (sCD163) has been associated with the M2 macrophage phenotype, and M2 macrophages have been shown to down-modulate inflammatory responses. In particular, previous studies have shown that M2 is closely associated with the most severe clinical presentation of leprosy (i.e. lepromatous leprosy (LL)), as well as tuberculosis. We hypothesized that sCD163 correlates with severity of diseases caused by intracellular pathogens. METHODOLOGY/PRINCIPAL FINDINGS: To assess this hypothesis, sCD163 levels were measured in the serum of leprosy and visceral leishmaniasis (VL) patients stratified by severity of the clinical presentation. sCD163 levels were significantly higher in patients with these diseases than those observed in healthy control individuals. Further analyses on infection and disease status of leprosy and VL patients revealed a clear association of sCD163 levels with clinical parameters of disease severity. In vitro culture assays revealed that Leishmania infection induced CD163 expression on the surface of both monocyte/macrophages and neutrophils, suggesting these cells as possible sources of sCD163. FACS analyses shows that the cells expressing CD163 produces both TNF-α and IL-4. CONCLUSIONS/SIGNIFICANCE: Taken together, our results reveal sCD163 as a potential biomarker of severity of diseases caused by intracellular pathogens M. leprae and Leishmania spp. and have a modulatory role, with a mix of an inflammatory property induced by TNF-α release, but that potentially induces an anti-inflammatory T cell response, related to IL-4 release.
Asunto(s)
Antígenos CD/sangre , Antígenos de Diferenciación Mielomonocítica/sangre , Biomarcadores/sangre , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/patología , Lepra/diagnóstico , Lepra/patología , Receptores de Superficie Celular/sangre , Humanos , Suero/química , Índice de Severidad de la EnfermedadRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Casearia sylvestris Sw. is widely used in popular medicine to treat conditions associated with pain. AIM OF THE STUDY: The present study investigated the influence of hydroalcoholic crude extract of Casearia sylvestris (HCE-CS) and contribution of pro-resolving mediators on mechanical hyperalgesia in a mouse model of chronic post-ischemia pain (CPIP). METHODS AND RESULTS: Male Swiss mice were subjected to ischemia of the right hind paw (3h), then reperfusion was allowed. At 10min, 24h or 48h post-ischemia/reperfusion (I/R), different groups of animals were treated with HCE-CS (30mg/Kg, orally [p.o]), selected agonists at the pro-resolving receptor ALX/FPR2 (natural molecules like resolvin D1 and lipoxin A4 or the synthetic compound BML-111; 0.1-1µg/animal) or vehicle (saline, 10mL/Kg, s.c.), in the absence or presence of the antagonist WRW4 (10µg, s.c.). Mechanical hyperalgesia (paw withdrawal to von Frey filament) was asseseed together with histological and immunostainning analyses. In these settings, pro-resolving mediators reduced mechanical hyperalgesia and HCE-CS or BML-111 displayed anti-hyperalgesic effects which was markedly attenuated in animals treated with WRW4. ALX/FPR2 expression was raised in skeletal muscle or neutrophils after treatment with HCE-CS or BML-111. CONCLUSION: These results reveal significant antihyperalgesic effect of HCE-CS on CPIP, mediated at least in part, by the pathway of resolution of inflammation centred on the axis modulated by ALX/FPR2.
Asunto(s)
Analgésicos/uso terapéutico , Casearia , Dolor Crónico/tratamiento farmacológico , Hiperalgesia/tratamiento farmacológico , Extractos Vegetales/uso terapéutico , Daño por Reperfusión/tratamiento farmacológico , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Analgésicos/farmacología , Animales , Anexina A1/genética , Dolor Crónico/metabolismo , Hiperalgesia/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Extractos Vegetales/farmacología , Hojas de la Planta , Receptores de Formil Péptido/metabolismo , Daño por Reperfusión/metabolismoRESUMEN
Omega-3 polyunsaturated fatty acids are essential for health and are known to possess anti-inflammatory properties, improving cardiovascular health as well as benefiting inflammatory diseases. Indeed, dietary supplementation with omega-3 polyunsaturated fatty acids has proved efficacious in reducing joint pain, morning stiffness and nonsteroidal anti-inflammatory drugs usage in rheumatoid arthritis patients. However, the mechanisms by which omega-3 polyunsaturated fatty acids exert their beneficial effects have not been fully explored. Seminal discoveries by Serhan and colleagues have unveiled a novel class of bioactive lipid mediators that are enzymatically biosynthesized in vivo from omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), termed resolvins, protectins and maresins. These bioactive pro-resolving lipid mediators provide further rationale for the beneficial effects of fish-oil enriched diets. These endogenous lipid mediators are spatiotemporally biosynthesized to actively regulate resolution by acting on specific G protein-coupled receptors (GPCRs) to initiate anti-inflammatory and pro-resolving signals that terminate inflammation. In this review, we will discuss the mechanism of actions of these molecules, including their analgesic and bone-sparing properties making them ideal therapeutic agonists for the treatment of inflammatory diseases such as rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Ácidos Docosahexaenoicos/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/metabolismo , Ácido Eicosapentaenoico/farmacología , Animales , Artritis Reumatoide/metabolismo , Progresión de la Enfermedad , Ácidos Docosahexaenoicos/uso terapéutico , Ácido Eicosapentaenoico/uso terapéutico , Humanos , Resultado del TratamientoRESUMEN
Microvesicles (MVs) are emerging as a new mechanism of intercellular communication by transferring cellular lipid and protein components to target cells, yet their function in disease is only now being explored. We found that neutrophil-derived MVs were increased in concentration in synovial fluid from rheumatoid arthritis patients compared to paired plasma. Synovial MVs overexpressed the proresolving, anti-inflammatory protein annexin A1 (AnxA1). Mice deficient in TMEM16F, a lipid scramblase required for microvesiculation, exhibited exacerbated cartilage damage when subjected to inflammatory arthritis. To determine the function of MVs in inflammatory arthritis, toward the possibility of MV-based therapeutics, we examined the role of immune cell-derived MVs in rodent models and in human primary chondrocytes. In vitro, exogenous neutrophil-derived AnxA1(+) MVs activated anabolic gene expression in chondrocytes, leading to extracellular matrix accumulation and cartilage protection through the reduction in stress-adaptive homeostatic mediators interleukin-8 and prostaglandin E2. In vivo, intra-articular injection of AnxA1(+) MV lessened cartilage degradation caused by inflammatory arthritis. Arthritic mice receiving adoptive transfer of whole neutrophils displayed abundant MVs within cartilage matrix and revealed that MVs, but not neutrophils themselves, can penetrate cartilage. Mechanistic studies support a model whereby MV-associated AnxA1 interacts with its receptor FPR2 (formyl peptide receptor 2)/ALX, increasing transforming growth factor-ß production by chondrocytes, ultimately leading to cartilage protection. We envisage that MVs, either directly or loaded with therapeutics, can be harnessed as a unique therapeutic strategy for protection in diseases associated with cartilage degeneration.
Asunto(s)
Artritis Reumatoide/patología , Cartílago Articular/patología , Neutrófilos/metabolismo , Animales , Humanos , Ratones , Líquido Sinovial/metabolismo , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Despite substantial efforts in recent years toward the development of new vaccines and drugs against tuberculosis (TB), success has remained elusive. Immunotherapy of TB with mycobacterial Hsp65 as a DNA vaccine (DNA-hsp65) results in a reduction of systemic bacterial loads and lung tissue damage, but the high homology of Hsp65 with the mammalian protein raises concern that pathological autoimmune responses may also be triggered. We searched for autoimmune responses elicited by DNA-hsp65 immunotherapy in mice chronically infected with TB by evaluating the humoral immune response and comprehensive histopathology using stereology. Cross-reactive antibodies between mycobacterial and mammalian Hsp60/65 were detected; however, no signs of pathological autoimmunity were found up to 60 days after the end of the therapy.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Autoinmunidad/inmunología , Proteínas Bacterianas/inmunología , Chaperonina 60/inmunología , Proteínas Mitocondriales/inmunología , Mycobacterium leprae/inmunología , Vacunas de ADN/inmunología , Animales , Autoinmunidad/efectos de los fármacos , Proteínas Bacterianas/administración & dosificación , Chaperonina 60/administración & dosificación , Chaperonina 60/antagonistas & inhibidores , Reacciones Cruzadas/efectos de los fármacos , Reacciones Cruzadas/inmunología , Inmunidad Humoral/efectos de los fármacos , Inmunidad Humoral/inmunología , Inmunoterapia/métodos , Ratones , Ratones Endogámicos BALB C , Proteínas Mitocondriales/antagonistas & inhibidores , Vacunas contra la Tuberculosis/administración & dosificación , Vacunas contra la Tuberculosis/inmunología , Vacunas de ADN/administración & dosificaciónRESUMEN
Despite the enormous efforts displayed globally in the fight against tuberculosis, the disease incidence has modified slightly, which has led to a renewed interest in immunotherapy. In general, successful immunotherapeutic candidates against tuberculosis are agents that can trigger strong, specific pro-inflammatory responses, especially of the T-helper (Th) 1 pattern. However, how these pro-inflammatory agents effectively kill the bacteria without eliciting immunopathology is not well understood. We reasoned that, in addition to the specific immune response elicited by immunotherapy, the evaluation of the overall pro-inflammatory responses should provide additional and valuable information that will be useful in avoiding immunopathology. We evaluated the overall IFN-γ and IL-17 pro-inflammatory responses among CD4(+), CD8(+) and γδ T cells in the lungs of mice that were infected with M. tuberculosis and treated with a DNA vaccine in an immunotherapeutic regimen. Our results demonstrate that mice that effectively combat the pathogen develop a strong, specific Th1 immune response against the therapeutic antigen and have reduced lung inflammation, present in parallel a fine-tuning in the total IFN-γ- and IL-17-mediated immunity in the lungs. This modulation of the total immune response involves reducing the Th17 cell population, augmenting CD8(+) T cells that produce IFN-γ and increasing the total γδ T cell frequency. These results stress the importance of a broad evaluation of not only the specific immune response at the time to evaluate new immune interventional strategies against tuberculosis but also non-conventional T cells, such as γδ T lymphocytes.