RESUMO
The functions of eicosanoids, a family of potent biologically active lipid mediators, are not restricted to inflammatory responses and they also act as mediators of the pathogenesis process. However, the role of eicosanoids in tuberculosis remains controversial. To investigate the specific role of LTB4 in Mycobacterium tuberculosis (Mtb) infection, we used 5-lipoxygenase-deficient (5-LO-/-) mice and WT (sv129) mice inoculated intranasally with LTB4 (encapsulated in PLGA microspheres). We showed that deficiency of the 5-LO pathway was related to resistance to Mtb infection. LTB4 inoculation increased susceptibility to Mtb in 5-LO-/- mice but not in WT mice, resulting in worsening of lung inflammation and tissue damage. In infected WT mice, most supplementary LTB4 was metabolized to the inactive form 12-oxo-LTB4 in the lung. A high amount of PGE2 was detected during Mtb infection, and pharmacological inhibition of COX-2 induced a significant reduction of bacterial load and an improved innate immune response in the lungs, independently of baseline LTB4 levels. COX-2 inhibition with celecoxib significantly reduced PGE2 levels, enhanced IFN-γ production and NO release, and increased macrophage phagocytosis of Mtb. The results suggest that a balance between PGE2/LTB4 is essential in the pathogenesis process of tuberculosis to prevent severe inflammation. Moreover, optimal levels of PGE2 are required to induce an effective innate response in the early phase of Mtb infection. Thus, pharmacological modulation of eicosanoid production may provide an important host-directed therapy in tuberculosis.
Assuntos
Dinoprostona/metabolismo , Eicosanoides/metabolismo , Inflamação/metabolismo , Leucotrieno B4/metabolismo , Metabolismo dos Lipídeos/fisiologia , Transdução de Sinais/fisiologia , Tuberculose/metabolismo , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Macrófagos/metabolismo , Masculino , CamundongosRESUMO
OBJECTIVE AND DESIGN: Investigate survival outcomes, and immunological and metabolomic effects of hyaluronidase (Hz) treatment during mouse models of acute inflammation and sepsis. METHODS: Survival of C57Bl/6 mice was monitored after lethal challenge with lipopolysaccharide (LPS) or cecal and ligation puncture (CLP)-induced sepsis and treated with Hz or saline. Mice were also challenged with LPS and treated with Hz for leukocyte counting, cytokine quantification and determination of metabolomic profiles in the peritoneal fluid. RESULTS: Hz treatment improved survival outcomes after lethal challenge with LPS or CLP-induced sepsis. LPS challenge promoted acute neutrophil accumulation and production of interleukin-1ß (IL-1ß) and IL-6 in the peritoneum, whereas Hz treatment suppressed neutrophil infiltration and cytokine production. We further characterized the metabolomic alterations caused by LPS challenge, which predicted activity of metabolic pathways related to fatty acids and eicosanoids. Hz treatment had a profound effect over the metabolic response, reflected by reductions of the relative levels of fatty acids. CONCLUSION: Collectively, these data demonstrate that Hz treatment is associated with metabolic reprogramming of pathways that sustain the inflammatory response.
Assuntos
Hialuronoglucosaminidase/farmacologia , Sepse/imunologia , Sepse/metabolismo , Doença Aguda , Animais , Líquido Ascítico/citologia , Líquido Ascítico/imunologia , Líquido Ascítico/metabolismo , Modelos Animais de Doenças , Eicosanoides/metabolismo , Ácidos Graxos/metabolismo , Imunomodulação , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Contagem de Leucócitos , Lipopolissacarídeos , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica , Camundongos Endogâmicos C57BLRESUMO
Needle-free immunization strategies have been sought for years. Transcutaneous immunization using electroporation has been studied, but the high electrical voltage that must be applied may be painful and cause irreversible cell damage. The application of a weak electric field, such as in iontophoresis, has never been attempted. The aim of this work was to verify the potential of employing iontophoresis for transcutaneous immunization using ovalbumin (OVA) as a model antigen. To target the antigen presenting cells that are located in the viable epidermis, a vaccine formulation composed of OVA-loaded liposomes and silver nanoparticles (NPAg) was developed. In vitro cathodal iontophoresis of the OVA-liposomes associated with NPAg increased OVA penetration into the viable epidermis by 92-fold in comparison to passive delivery. In vivo, transcutaneous immunization with a suitable combination of liposome and iontophoresis induced the production of antibodies, differentiation of immune-competent cells and appeared to present an alternative strategy for needle-free vaccination.
Assuntos
Imunização/métodos , Iontoforese , Nanopartículas , Administração Cutânea , Ovalbumina , VacinaçãoRESUMO
Microencapsulation of bioactive molecules for modulating the immune response during infectious or inflammatory events is a promising approach, since microspheres (MS) protect these labile biomolecules against fast degradation, prolong the delivery over longer periods of time and, in many situations, target their delivery to site of action, avoiding toxic side effects. Little is known, however, about the influence of different polymers used to prepare MS on macrophages. This paper aims to address this issue by evaluating in vitro cytotoxicity, phagocytosis profile and cytokines release from alveolar macrophages (J-774.1) treated with MS prepared with chitosan, and four different co-polymers of PLGA [poly (lactic-co-glycolic acid)]. The five MS prepared presented similar diameter and zeta potential each other. Chitosan-MS showed to be cytotoxic to J-774.1 cells, in contrast to PLGA-MS, which were all innocuous to this cell linage. PLGA 5000-MS was more efficiently phagocytized by macrophages compared to the other MS tested. PLGA 5000-MS and 5002-MS induced significant production of TNF-α, while 5000-MS, 5004-MS and 7502-MS decreased spontaneous IL-6 release. Nevertheless, only PLGA 5002-MS induced significant NFkB/SEAP activation. These findings together show that MS prepared with distinct PLGA co-polymers are differently recognized by macrophages, depending on proportion of lactic and glycolic acid in polymeric chain, and on molecular weight of the co-polymer used. Selection of the most adequate polymer to prepare a microparticulate drug delivery system to modulate immunologic system may take into account, therefore, which kind of immunomodulatory response is more adequate for the required treatment.
Assuntos
Quitosana/química , Ácido Láctico/química , Macrófagos/efeitos dos fármacos , Microesferas , Ácido Poliglicólico/química , Polímeros/farmacologia , Microscopia Eletrônica de Varredura , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
Biodegradable lactic-co-glycolic acid (PLGA) microspheres (MS) improve the stability of biomolecules stability and allow enable their sustained release. Lipid mediators represent a strategy for improving host defense; however, most of these mediators, such as prostaglandin D2 (PGD2), have low water solubility and are unstable. The present study aimed to develop and characterize MS loaded with PGD2 (PGD2-MS) to obtain an innovative tool to activate macrophages. PGD2-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process, and the size, zeta potential, surface morphology and encapsulation efficiency were determined. It was also evaluated in vitro the phagocytic index, NF-κB activation, as well as nitric oxide and cytokine production by alveolar macrophages (AMs) in response to PGD2-MS. PGD2-MS were spherical with a diameter of 5.0±3.3 µm and regular surface, zeta potential of -13.4±5.6 mV, and 36% of encapsulation efficiency, with 16-26% release of entrapped PGD2 at 4 and 48 h, respectively. PGD2-MS were more efficiently internalized by AMs than unloaded-MS, and activated NF-κB more than free PGD2. Moreover, PGD2-MS stimulated the production of nitric oxide, TNF-α, IL-1ß, and TGF-ß, more than free PGD2, indicating that microencapsulation increased the activating effect of PGD2 on cells. In LPS-pre-treated AMs, PGD2-MS decreased the release of IL-6 but increased the production of nitric oxide and IL-1ß. These results show that the morphological characteristics of PGD2-MS facilitated interaction with, and activation of phagocytic cells; moreover, PGD2-MS retained the biological activities of PGD2 to trigger effector mechanisms in AMs. It is suggested that PGD2-MS represent a strategy for therapeutic intervention in the lungs of immunocompromised subjects.
Assuntos
Macrófagos Alveolares/efeitos dos fármacos , Microesferas , Prostaglandina D2/administração & dosagem , Animais , Linhagem Celular , Células Cultivadas , Liberação Controlada de Fármacos , Emulsões , Interleucina-1beta/metabolismo , Ácido Láctico/química , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/fisiologia , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Tamanho da Partícula , Fagocitose , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Prostaglandina D2/química , Prostaglandina D2/farmacologia , Ratos Wistar , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The aim of this work was to develop an innovative tool for the treatment of pulmonary fibrosis based on our previous findings, which demonstrated that intranasally administered soluble bovine hyaluronidase (HYAL) increases the numbers of mesenchymal (MSC)-like cells in the bronchoalveolar fluid (BALF) and thus reduces the bleomycin-induced fibrosis. To this end, we developed poly(D,L-lactide-co-glycolide) (PLGA) microparticles (MPs) loaded with HYAL (HYAL-MP) to preserve the enzyme's biological activity and to facilitate its delivery to the lung. Nonloaded MPs (Control-MPs) and HYAL-MPs were prepared using the emulsion and solvent evaporation methods and thoroughly characterized. The HYAL-MPs and Control-MPs exhibited an average diameter of 4.3±2.1 and 4.4±1.5 µm, respectively. The encapsulation efficiency of the HYAL-MPs was 68%, and encapsulation led to a reduced release rate. Additionally, the HYAL-MPs were efficiently phagocytosed by J-774.1 cells. Compared with the soluble HYAL, the HYAL-MPs increased the proportion of MSC-like cells in the BALF of C57BL6 mice 96 h after treatment. The efficacy of the HYAL-MPs was also tested in C57BL6 mice that were previously exposed to 4 U/kg of bleomycin to induce lung fibrosis. The results demonstrated that the HYAL-MPs reduced neutrophil recruitment after bleomycin treatment more effectively than did the soluble HYAL, whereas the Control-MPs did not exhibit any effect. The HYAL-MPs also reduced the bleomycin-induced fibrosis more efficiently, and 134% of the collagen deposition in the lung compared with the soluble HYAL and the Control-MPs. In summary, our data indicate that HYAL-MPs are an effective delivery system that could feasibly be used in the treatment of pulmonary fibrosis.
Assuntos
Hialuronoglucosaminidase/uso terapêutico , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Fibrose Pulmonar/tratamento farmacológico , Animais , Bovinos , Contagem de Células , Linhagem Celular , Colágeno/metabolismo , Citoesqueleto/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Fagócitos/citologia , Pneumonia/complicações , Pneumonia/tratamento farmacológico , Pneumonia/patologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Fibrose Pulmonar/complicações , Fibrose Pulmonar/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solubilidade , Eletricidade Estática , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix. They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids. Here, we investigated the influence of bovine testes hyaluronidase (HYAL) during cutaneous wound healing in in vitro and in vivo assays. We demonstrated in the wound scratch assay that HYAL increased the migration and proliferation of fibroblasts in vitro at low concentration, e.g. 0.1 U HYAL enhanced the cell number by 20%. HYAL presented faster and higher reepithelialization in in vivo full-thickness excisional wounds generated on adult Wistar rats back skin already in the early phase at 2nd day post operatory compared to vehicle-control group. Wound closured area observed in the 16 U and 32 U HYAL treated rats reached 38% and 46% compared to 19% in the controls, respectively. Histological and biochemical analyses supported the clinical observations and showed that HYAL treated wounds exhibited increased granulation tissue, diminished edema formation and regulated the inflammatory response by modulating the release of pro and anti-inflammatory cytokines, growth factor and eicosanoids mediators. Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR ß/δ, the collagen content in the early stages of healing processes as well as angiogenesis. Altogether these data revealed that HYAL accelerates wound healing processes and might be beneficial for treating wound disorders.
Assuntos
Fibroblastos/fisiologia , Hialuronoglucosaminidase/farmacologia , Inflamação/imunologia , Fenômenos Fisiológicos da Pele , Cicatrização/fisiologia , Animais , Movimento Celular , Proliferação de Células , Colágeno/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Eicosanoides/metabolismo , Fibroblastos/efeitos dos fármacos , Tecido de Granulação/efeitos dos fármacos , Tecido de Granulação/crescimento & desenvolvimento , Masculino , Camundongos , Receptores Ativados por Proliferador de Peroxissomo/efeitos dos fármacos , Ratos , Ratos Wistar , Cicatrização/efeitos dos fármacosRESUMO
Scorpion sting-induced human envenomation provokes an intense inflammatory reaction. However, the mechanisms behind the recognition of scorpion venom and the induction of mediator release in mammalian cells are unknown. We demonstrated that TLR2, TLR4 and CD14 receptors sense Tityus serrulatus venom (TsV) and its major component, toxin 1 (Ts1), to mediate cytokine and lipid mediator production. Additionally, we demonstrated that TsV induces TLR2- and TLR4/MyD88-dependent NF-κB activation and TLR4-dependent and TLR2/MyD88-independent c-Jun activation. Similar to TsV, Ts1 induces MyD88-dependent NF-κB phosphorylation via TLR2 and TLR4 receptors, while c-Jun activation is dependent on neither TLR2 nor TLR4/MyD88. Therefore, we propose the term venom-associated molecular pattern (VAMP) to refer to molecules that are introduced into the host by stings and are recognized by PRRs, resulting in inflammation.
Assuntos
Mediadores da Inflamação/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos Peritoneais/metabolismo , Venenos de Escorpião/farmacologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Receptores de Lipopolissacarídeos/genética , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genéticaRESUMO
Insufficient oxygen delivery to organs leads to tissue dysfunction and cell death. Reperfusion, although vital to organ survival, initiates an inflammatory response that may both aggravate local tissue injury and elicit remote organ damage. Polymorphonuclear neutrophil (PMN) trafficking to remote organs following ischaemia/reperfusion (I/R) is associated with the release of lipid mediators, including leucotriene (LT) B4 , cysteinyl-LTs (CysLTs) and platelet-activating factor (PAF). Yet, their potentially cooperative role in regulating I/R-mediated inflammation has not been thoroughly assessed. The present study aimed to determine the cooperative role of lipid mediators in regulating PMN migration, tissue oedema and injury using selective receptor antagonists in selected models of I/R and dermal inflammation. Our results show that rabbits, pre-treated orally with BIIL 284 and/or WEB 2086 and MK-0571, were protected from remote tissue injury following I/R or dermal inflammation in an additive or synergistic manner when the animals were pre-treated with two drugs concomitantly. The functional selectivity of the antagonists towards their respective agonists was assessed in vitro, showing that neither BIIL 284 nor WEB 2086 prevented the inflammatory response to IL-8, C5a and zymosan-activated plasma stimulation. However, these agonists elicited LTB4 biosynthesis in isolated rabbit PMNs. Similarly, a cardioprotective effect of PAF and LTB4 receptor antagonists was shown following myocardial I/R in mice. Taken together, these results underscore the intricate involvement of LTB4 and PAF in each other's responses and provide further evidence that targeting both LTs and PAF receptors provides a much stronger anti-inflammatory effect, regulating PMN migration and oedema formation.
Assuntos
Leucotrienos/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Amidinas/farmacologia , Animais , Azepinas/farmacologia , Bioensaio , Carbamatos/farmacologia , Derme/patologia , Modelos Animais de Doenças , Extravasamento de Materiais Terapêuticos e Diagnósticos/metabolismo , Extravasamento de Materiais Terapêuticos e Diagnósticos/patologia , Extremidades/irrigação sanguínea , Extremidades/patologia , Inflamação/patologia , Leucotrieno B4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Propionatos/farmacologia , Quinolinas/farmacologia , Coelhos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Leucotrienos/agonistas , Receptores de Leucotrienos/metabolismo , Triazóis/farmacologiaRESUMO
Prostaglandins act as mediators of inflammation and, similar to cytokines, function as immune modulators during innate and adaptive immune responses. Therefore, using a pharmacological inhibitor, celecoxib, we investigated the role of prostaglandins in host defense against Histoplasma capsulatum infection in C57BL/6 mice. Our results showed that treatment with celecoxib inhibited cyclooxygenase 2, reduced the total fungal burden, and reduced the concentration of PGE2, cytokines, lymphocytes, neutrophils, and mononuclear cells in the bronchoalveolar space and lung parenchyma. In addition, celecoxib treatment increased the synthesis of nitric oxide, IFN- γ, LTB4, and the phagocytic capacity of alveolar macrophages. Moreover, celecoxib treatment increased the survival of mice after infection with a lethal inoculum of H. capsulatum. These results suggest that prostaglandins alter the host immune response and play an important role in the pathogenesis of histoplasmosis. Thus, the inhibition of prostaglandins could be a valuable immunomodulatory strategy and antifungal therapy for histoplasmosis treatment.