RESUMO
Increasing attention is given to the finding that macrophages under hypoxia are capable of controlling infection by the intracellular protozoan parasite Leishmania amazonensis. The hypoxia-inducible factor (HIF)-1α has been shown to play an essential role in this enhanced innate immune response. Our study aimed to explore the HIF-1α-dependent mechanisms leading to reduced survival of the parasites residing in macrophages under low oxygen conditions. Hypoxia triggered (Pâ¯<â¯0.01) NADPH oxidase 2 (Nox2) expression and reactive oxygen species (ROS) production in J774 macrophages upon 24-h infection with L. amazonensis. Furthermore, increased (Pâ¯<â¯0.01) expression levels of HIF-1α and macrophage migration inhibitory factor (MIF) were detected in the infected cells grown at 3% oxygen tension. We found that either HIF-1α silencing, Nox2 inhibition or MIF antagonism caused a significant (Pâ¯<â¯0.05) reversal of the improved leishmanicidal activity displayed by the hypoxic phagocytes. Taken together, our current results suggest that, under conditions of limited availability of oxygen, activation of the HIF-1α/MIF axis via Nox2/ROS induction promotes killing of L. amazonensis amastigotes by macrophages. Such protective mechanism might operate in L. amazonensis-infected tissues where low oxygen levels prevail.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Macrófagos/imunologia , Animais , Hipóxia Celular , Linhagem Celular , Hipóxia/imunologia , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imunidade Inata , Oxirredutases Intramoleculares/fisiologia , Leishmania/imunologia , Leishmania/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NADPH Oxidase 2/metabolismo , NADPH Oxidase 2/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Macrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy. METHODS: Mif was immunolocalized at implantation sites on gestation days (gd) 7.5, 10.5, 13.5 and 17.5. Ectoplacental cones and fetal placentas dissected from the maternal tissues were used for Western blotting and qRT-PCR assays on the same gestation days. RESULTS: During the post-implantation period (gd7.5), trophoblast giant cells showed strong Mif reactivity. In later placentation phases (gds 10.5-17.5), Mif appeared to be concentrated in the junctional zone and trophoblast giant cells. Mif protein expression increased significantly from gd7.5 to 10.5 (p = 0.005) and from gd7.5 to 13.5 (p = 0.03), remaining at high concentration as gestation proceeded. Higher mRNA expression was found on gd10.5 and was significantly different from gd13.5 (p = 0.048) and 17.5 (p = 0.009). CONCLUSIONS: The up-regulation of Mif on gd10.5 coincides with the stage in which the placenta assumes its three-layered organization (giant cells, spongiotrophoblast and labyrinth zones), fetal blood circulation begins and population of uNK cells reaches high proportions at the maternal counter part of the placenta, suggesting that Mif may play a role in either the placentation or in the adaptation of the differentiated placenta to the uterus or still in gestational immunomodulatory responses. Moreover, it reinforces the possibility of specific activities for Mif at the maternal fetal interface.
Assuntos
Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Placenta/metabolismo , Animais , Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Feminino , Regulação da Expressão Gênica , Idade Gestacional , Imuno-Histoquímica , Oxirredutases Intramoleculares/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Camundongos , Reação em Cadeia da Polimerase , Gravidez , Distribuição TecidualRESUMO
Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of MIF-dependent responses to Toxoplasma gondii, we investigated host resistance in MIF-/- mice (BALB/c background) during natural oral infection. We focused on the potential involvement of MIF in Dendritic Cell (DC) maturation and IL-12 production. Following oral T. gondii infection, wild type mice developed a strong IL-12 response with an adequate maturation of their draining mesenteric lymph node DC (MLNDC) population and were resistant to challenge with either 40 or 100 cysts (ME49 strain). In contrast, similarly infected MIF-/- mice mounted a weak IL-12 response, displayed immature MLNDCs in the early phases of infection and rapidly succumbed to both type of challenges. Lack of maturation and IL-12 production of DCs in response to T. gondii antigens was confirmed by in vitro studies, and these effects were reversed following treatment with recombinant MIF. These findings demonstrate that MIF-induced early DC maturation and IL-12 production mediate resistance to T. gondii infection.
Assuntos
Células Dendríticas/patologia , Interleucina-12/biossíntese , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Toxoplasmose Animal/imunologia , Animais , Antígenos de Protozoários/imunologia , Encéfalo/parasitologia , Células Dendríticas/imunologia , Progressão da Doença , Suscetibilidade a Doenças/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Oxirredutases Intramoleculares/fisiologia , Fígado/parasitologia , Fígado/patologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organismos Livres de Patógenos Específicos , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/imunologia , Regulação para CimaRESUMO
Macrophage migration inhibitory factor (MIF) exerts either a protective or a deleterious role in the immune response to different pathogens. We analyzed herein the role of MIF in the host control of toxoplasmosis using MIF(-/-) mice backcrossed to either the BALB/c or the C57BL/6 genetic backgrounds. Both, wild-type (WT) BALB/c and MIF(-/-) BALB/c mice were susceptible to infection with highly virulent RH as well as moderately virulent ME49 strains of T. gondii. MIF(-/-) mice, however, showed greater liver damage and more brain cysts, produced less proinflammatory cytokines, and succumbed significantly faster than WT mice. Bone marrow-derived dendritic cells (BMDCs) from MIF(-/-) mice produced less interleukin-1beta, interleukin-12, and tumor necrosis factor-alpha than WT BMDCs after stimulation with soluble Toxoplasma antigen (STAg). Similar observations were made in CD11c(+) low-density cells isolated from the spleens of MIF(-/-) mice challenged with STAg. MIF(-/-) C57BL/6 mice succumbed to ME49 infection faster than their WT counterparts. C57BL/6 mice that succumbed to infection with the ME49 strain produced less MIF than resistant BALB/c mice similarly infected. Interestingly, an analysis of brains from patients with cerebral toxoplasmosis showed low levels of MIF expression. Together, these findings demonstrate that MIF plays a critical role in mediating host resistance against T. gondii.
Assuntos
Predisposição Genética para Doença , Interações Hospedeiro-Parasita , Imunidade Inata , Oxirredutases Intramoleculares/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Toxoplasmose/genética , Animais , Encéfalo/metabolismo , Encéfalo/parasitologia , Citocinas/biossíntese , Células Dendríticas/imunologia , Encefalite/imunologia , Encefalite/parasitologia , Encefalite/patologia , Hepatite/imunologia , Hepatite/parasitologia , Hepatite/patologia , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Imunidade Inata/genética , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Camundongos , Camundongos Mutantes , Ácido Nítrico/metabolismo , Receptor 2 Toll-Like/biossíntese , Toxoplasma/patogenicidade , Toxoplasmose/imunologia , Toxoplasmose/patologia , Toxoplasmose Cerebral/imunologia , Toxoplasmose Cerebral/parasitologia , Toxoplasmose Cerebral/patologia , VirulênciaRESUMO
The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO(2) in air) for 1h in the following treatments either 500 microL of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1h either FM or FM with alpha L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) alpha L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) alpha L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 x 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha L-PGDS: (1) 26.4+/-3.0; (2) 25.6+/-3.0; (3) 59.7+/-3.0; (4) 56.4+/-3.0; and (5) 57.1+/-3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P<0.05) compared with the control: (1) 89.2+/-2.0%; (2) 87.5+/-2.0%; (3) 19.4+/-2.0%; (4) 27.2+/-3.1%; and (5) 14.1+/-3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization.
Assuntos
Anticorpos/farmacologia , Bovinos/fisiologia , Fertilização in vitro/veterinária , Oxirredutases Intramoleculares/imunologia , Lipocalinas/imunologia , Oócitos/efeitos dos fármacos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Feminino , Fertilização in vitro/efeitos dos fármacos , Imunoglobulina G/farmacologia , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/fisiologia , Lipocalinas/química , Lipocalinas/fisiologia , Masculino , Fatores de TempoRESUMO
Macrophage migration inhibitory factor (MIF) is increased in asthmatic patients and plays a critical role in the pathogenesis of asthma. We show here that mice lacking MIF failed to develop airway hyper-responsiveness (AHR), tissue eosinophilia, and mucus metaplasia. Analysis of the bronchoalveolar fluids revealed a substantial reduction of IL-13, eotaxin and cysteinyl-leukotrienes. The lack of these cardinal features of asthma in MIF(-/-) mice occurs regardless of high concentrations of IL-4 in the lung and OVA-specific IgE in the serum. Antigen-specific lymphocyte proliferation and IL-13 production were similarly increased in the draining lymph nodes of OVA-immunized and challenged MIF(-/-) mice compared to WT, but were reduced in the spleen of MIF(-/-), thus indicating differential roles of MIF in these compartments. Stimulation of naive CD4(+) cells with anti-CD3 antibody demonstrated that MIF(-/-) cells produced increased amounts of IFN-gamma and IL-4 compared to WT CD4(+) cells. Finally, treatment of sensitized BALB/c mice with neutralizing anti-MIF antibody abrogated the development of ARH and airway inflammation without affecting the production of Th2 cytokines or IgE. The present study demonstrates that MIF is required for allergic inflammation, adding important elements to our knowledge of asthma pathogenesis and suggesting that neutralization of MIF might be of therapeutic value in asthma.