RESUMO
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
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
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.
Assuntos
Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Histoplasma/patogenicidade , Histoplasmose/tratamento farmacológico , Histoplasmose/metabolismo , Pirazóis/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Celecoxib , Histoplasma/efeitos dos fármacos , Interferon gama/metabolismo , Leucotrieno B4/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismoRESUMO
Inflammatory mediators are thought to be involved in the systemic and local immune response induced by the Tityus serrulatus scorpion envenomation. New functional aspects of lipid mediators have recently been described. Here, we examine the unreported role of lipid mediators in cell recruitment to the peritoneal cavity after an injection with Ts2 or Ts6 toxins isolated from the T. serrulatus scorpion venom. In this report, we demonstrate that following a single intraperitoneal (i.p.) injection of Ts2 or Ts6 (250 µg/kg) in mice, there was an induction of leukocytosis with a predominance of neutrophils observed at 4, 24, 48 and 96 h. Moreover, total protein, leukotriene (LT)B(4), prostaglandin (PG)E(2) and pro-inflammatory cytokine levels were increased. We also observed an increase of regulatory cytokines, including interleukin (IL)-10, after the Ts2 injection. Finally, we observed that Ts2 or Ts6 injection in 5-lipoxygenase (LO) deficient mice and in wild type (WT) 129sv mice pre-treated with LTs and PGs inhibitors (MK-886 and celecoxib, respectively) a reduction the influx of leukocytes occurs in comparison to WT. The recruitment of these cells demonstrated a phenotype characteristic of neutrophils, macrophages, CD4 and CD8 lymphocytes expressing GR1+, F4/80+, CD3+/CD4+ and CD3+/CD8+, respectively. In conclusion, our data demonstrate that Ts2 and Ts6 induce inflammation by mechanisms dependent on lipid mediators and cytokine production. Ts2 may play a regulatory role whereas Ts6 exhibits pro-inflammatory activity exclusively.
Assuntos
Citocinas/biossíntese , Mediadores da Inflamação/fisiologia , Inflamação/induzido quimicamente , Lipídeos/fisiologia , Venenos de Escorpião/toxicidade , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Celecoxib , Movimento Celular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Imuno-Histoquímica , Indóis/farmacologia , Inflamação/patologia , Leucócitos/efeitos dos fármacos , Leucotrienos/biossíntese , Leucotrienos/fisiologia , Inibidores de Lipoxigenase/farmacologia , Masculino , Camundongos , Cavidade Peritoneal/patologia , Prostaglandinas/biossíntese , Prostaglandinas/fisiologia , Pirazóis/farmacologia , Sulfonamidas/farmacologiaRESUMO
Scorpion envenomation induces a systemic immune response, and neurotoxins of venom act on specific ion channels, modulating neurotransmitter release or activity. However, little is known about the immunomodulatory effects of crude venom from scorpion Tityus serrulatus (TsV) or its toxins (Ts1, Ts2 and Ts6) in combination with lipopolysaccharide (LPS). To investigate the immunomodulatory effects of TsV and its toxins (Ts1, Ts2 and Ts6), J774.1 cells were stimulated with different concentrations (25, 50 and 100 µg/mL) of venom or toxins pre-stimulated or not with LPS (0.5 µg/mL). Macrophage cytotoxicity was assessed, and nitric oxide (NO) and cytokine production were analyzed utilizing the culture supernatants. TsV and its toxins did not produce cytotoxic effects. Depending on the concentrations used, TsV, Ts1 and Ts6 stimulated the production of NO, interleukin (IL)-6 and tumor necrosis factor (TNF)-α in J774.1 cells, which were enhanced under LPS co-stimulation. However, LPS + Ts2 inhibited NO, IL-6 and TNF-α production, and Ts2 alone stimulated the production of IL-10, suggesting an anti-inflammatory activity for this toxin. Our findings are important for the basic understanding of the mechanisms involved in macrophage activation following envenomation; additionally, these findings may contribute to the discovery of new therapeutic compounds to treat immune-mediated diseases.