RESUMO
BACKGROUND: Subcutaneous implants of heat-coagulated egg white (egg white implants, EWI) induce intense local eosinophilia and prime for hyperreactivity following airway ovalbumin challenge. The roles of allergen sensitization, surgical trauma-induced glucocorticoids, and the 5-lipoxygenase (5-LO) pathway were hitherto unexplored in this model, in which quantitative recovery and large-scale purification of the eosinophils from the inflammatory site for functional and immunopharmacological studies are difficult to achieve. METHODS: We overcame this limitation by shifting the implantation site to the peritoneal cavity (EWIp), thereby enabling quantitative leukocyte retrieval. RESULTS: By day 7 post-surgery, eosinophil counts reached ~ 30% of all leukocytes recovered. Eosinophilia was prevented by: a) induction of allergen-specific oral tolerance to ovalbumin, the main allergen in egg white; b) inactivation of the 5-lipoxygenase pathway; c) blockade of endogenous glucocorticoid signaling by pretreatment with metirapone plus mifepristone before surgery. Highly purified eosinophils (~99% pure) could be obtained from the peritoneal exudate of EWIp-carrier mice in 2 simple, antibody-free steps. Preparative-scale yields, suitable for most biochemical, pharmacological, and molecular applications, were routinely obtained, and could be further enhanced through addition of pre-or post-surgery immunization steps (active or adoptive). The recovered eosinophils were fully functional in vivo, as demonstrated by the transfer of purified eosinophils into eosinophil-deficient Δdbl-GATA-1-KO mice, which upon subsequent challenge with eotaxin-1 present secondary accumulation of neutrophils, but not of mononuclear phagocytes. CONCLUSION: These findings document glucocorticoid-, allergen- and 5-lipoxygenase-dependent eosinophilia, which makes EWIp carriers an abundant source of pure, nontransgenic eosinophils for immunopharmacological studies.
Assuntos
Alérgenos/imunologia , Araquidonato 5-Lipoxigenase/imunologia , Eosinofilia/imunologia , Glucocorticoides/imunologia , Ovalbumina/imunologia , Animais , Araquidonato 5-Lipoxigenase/genética , Eosinófilos/imunologia , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos AnimaisRESUMO
Brucella abortus is a Gram-negative bacterium that infects humans and cattle, causing a chronic inflammatory disease known as brucellosis. A Th1-mediated immune response plays a critical role in host control of this pathogen. Recent findings indicate contrasting roles for lipid mediators in host responses against infections. 5-Lipoxygenase (5-LO) is an enzyme required for the production of the lipid mediators leukotrienes and lipoxins. To determine the involvement of 5-LO in host responses to B. abortus infection, we intraperitoneally infected wild-type and 5-LO-deficient mice and evaluated the progression of infection and concomitant expression of immune mediators. Here, we demonstrate that B. abortus induced the upregulation of 5-LO mRNA in wild-type mice. Moreover, this pathogen upregulated the production of the lipid mediators leukotriene B4 and lipoxin A4 in a 5-LO-dependent manner. 5-LO-deficient mice displayed lower bacterial burdens in the spleen and liver and less severe liver pathology, demonstrating an enhanced resistance to infection. Host resistance paralleled an increased expression of the proinflammatory mediators interleukin-12 (IL-12), gamma interferon (IFN-γ), and inducible nitric oxide synthase (iNOS) during the course of infection. Moreover, we demonstrated that 5-LO downregulated the expression of IL-12 in macrophages during B. abortus infection. Our results suggest that 5-LO has a major involvement in B. abortus infection, by functioning as a negative regulator of the protective Th1 immune responses against this pathogen.
Assuntos
Araquidonato 5-Lipoxigenase/imunologia , Brucella abortus/imunologia , Brucelose/enzimologia , Brucelose/imunologia , Células Th1/imunologia , Animais , Araquidonato 5-Lipoxigenase/deficiência , Araquidonato 5-Lipoxigenase/genética , Carga Bacteriana , Brucelose/microbiologia , Brucelose/patologia , Progressão da Doença , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Imunidade Inata , Injeções Intraperitoneais , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Leucotrieno B4/biossíntese , Lipoxinas/biossíntese , Fígado/imunologia , Fígado/microbiologia , Fígado/patologia , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Baço/imunologia , Baço/microbiologia , Baço/patologia , Células Th1/microbiologia , Células Th1/patologiaRESUMO
The bioactive lipid mediator leukotriene B4 (LTB4) greatly enhances phagocyte antimicrobial functions against a myriad of pathogens. In murine histoplasmosis, inhibition of the LT-generating enzyme 5-lypoxigenase (5-LO) increases the susceptibility of the host to infection. In this study, we investigated whether murine resistance or susceptibility to Histoplasma capsulatum infection is associated with leukotriene production and an enhancement of in vivo and/or in vitro antimicrobial effector function. We show that susceptible C57BL/6 mice exhibit a higher fungal burden in the lung and spleen, increased mortality, lower expression levels of 5-LO and leukotriene B4 receptor 1 (BLT1) and decreased LTB4 production compared to the resistant 129/Sv mice. Moreover, we demonstrate that endogenous and exogenous LTs are required for the optimal phagocytosis of H. capsulatum by macrophages from both murine strains, although C57BL/6 macrophages are more sensitive to the effects of LTB4 than 129/Sv macrophages. Therefore, our results provide novel evidence that LTB4 production and BLT1 signaling are required for a histoplasmosis-resistant phenotype.
Assuntos
Histoplasma/imunologia , Histoplasmose/veterinária , Leucotrieno B4 , Receptores do Leucotrieno B4/imunologia , Doenças dos Roedores/imunologia , Animais , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/imunologia , Suscetibilidade a Doenças , Inibidores Enzimáticos/farmacologia , Expressão Gênica/imunologia , Histoplasma/patogenicidade , Histoplasmose/genética , Histoplasmose/imunologia , Histoplasmose/metabolismo , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Leucotrieno B4/metabolismo , Leucotrieno B4/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Fagocitose/efeitos dos fármacos , Receptores do Leucotrieno B4/genética , Doenças dos Roedores/genética , Doenças dos Roedores/metabolismo , Transdução de Sinais , Baço/efeitos dos fármacos , Baço/imunologia , Baço/microbiologiaRESUMO
Lipid mediators derived from 5-lipoxygenase (5-LO) metabolism can activate both pro- and anti-inflammatory pathways, but their role in wound healing remains largely unexplored. In this study we show that 5-LO knockout (5-LO(-/-)) mice exhibited faster wound healing than wild-type (WT) animals, and exhibited upregulation of heme oxygenase-1 (HO-1). Furthermore, HO-1 inhibition in 5-LO(-/-) mice abolished the beneficial effect observed. Despite the fact that 5-LO(-/-) mice exhibited faster healing, in in vitro assays both migration and proliferation of human dermal fibroblasts (HDFs) were inhibited by the 5-LO pharmacologic inhibitor AA861. No changes were observed in the expression of fibronectin, transforming growth factor (I and III), and α-smooth muscle actin (α-SMA). Interestingly, AA861 treatment significantly decreased ROS formation by stimulated fibroblasts. Similar to 5-LO(-/-) mice, induction of HO-1, but not superoxide dismutase-2 (SOD-2), was also observed in response to 5-LO (AA861) or 5-LO activating protein (MK886) inhibitors. HO-1 induction was independent of nuclear factor (erythroid derived-2) like2 (Nrf-2), cyclooxygenase 2 (COX-2) products, or lipoxin action. Taken together, our results show that 5-LO disruption improves wound healing and alters fibroblast function by an antioxidant mechanism based on HO-1 induction. Overexpression of HO-1 in wounds may facilitate early wound resolution.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Dermatite/metabolismo , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Cicatrização/fisiologia , Adulto , Animais , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/imunologia , Dermatite/genética , Dermatite/imunologia , Derme/citologia , Derme/imunologia , Derme/metabolismo , Modelos Animais de Doenças , Fibroblastos/imunologia , Fibroblastos/metabolismo , Heme Oxigenase-1/imunologia , Humanos , Masculino , Proteínas de Membrana/imunologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Estresse Oxidativo/fisiologia , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismoRESUMO
This study provides evidence supporting the idea that although inflammatory cells migration to the cardiac tissue is necessary to control the growth of Trypanosoma cruzi, the excessive influx of such cells during acute myocarditis may be deleterious to the host. Production of lipid mediators of inflammation like leukotrienes (LTs) along with cytokines and chemokines largely influences the severity of inflammatory injury in response to tissue parasitism. T. cruzi infection in mice deficient in 5-lipoxygenase (5-LO), the enzyme responsible for the synthesis of LTs and other lipid inflammatory mediators, resulted in transiently increased parasitemia, and improved survival rate compared with WT mice. Myocardia from 5-LO(-/-) mice exhibited reduced inflammation, collagen deposition, and migration of CD4(+), CD8(+), and IFN-gamma-producer cells compared with WT littermates. Moreover, decreased amounts of TNF-alpha, IFN-gamma, and nitric oxide synthase were found in the hearts of 5-LO(-/-) mice. Interestingly, despite of early higher parasitic load, 5-LO(-/-) mice survived, and controlled T. cruzi infection. These results show that efficient parasite clearance is possible in a context of moderate inflammatory response, as occurred in 5-LO(-/-) mice, in which reduced myocarditis protects the animals during T. cruzi infection.
Assuntos
Araquidonato 5-Lipoxigenase/imunologia , Araquidonato 5-Lipoxigenase/metabolismo , Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/parasitologia , Inflamação/patologia , Trypanosoma cruzi/patogenicidade , Animais , Araquidonato 5-Lipoxigenase/deficiência , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/mortalidade , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Leucotrienos/imunologia , Leucotrienos/metabolismo , Camundongos , Camundongos Knockout , Miocárdio/patologia , Parasitemia/imunologia , Análise de Sobrevida , Trypanosoma cruzi/imunologiaRESUMO
Herein, we investigated the involvement of the 5-LO-derived lipid mediator LTB(4) in gammadelta T cell migration. When injected into the i.pl. space of C57BL/6 mice, LTB(4) triggered gammadelta T lymphocyte mobilization in vivo, a phenomenon also observed in in vitro chemotaxis assays. The i.pl. injection of Escherichia coli endotoxin (LPS) triggered increased levels of LTB(4) in pleural cavities. The in vivo inhibition of LTB(4) biosynthesis by the 5-LO inhibitor zileuton or the FLAP inhibitor MK886 attenuated LPS-induced gammadelta T cell accumulation into pleural cavities. Accordingly, 5-LO KO mice failed to recruit gammadelta T cells into the inflammatory site after i.pl. LPS. Antagonists of the high-affinity LTB(4) receptor BLT1, CP105,696, and LY292476 also attenuated LPS-induced gammadelta T cell accumulation in pleural cavities as well as in vitro chemotaxis toward pleural washes obtained from LPS-simulated mice. LTB(4)/BLT1 also accounted for gammadelta T cell migration induced by i.pl. administration of Mycobacterium bovis BCG or antigen in sensitized mice. BLT1 was expressed on naïve, resident as well as LPS-recruited gammadelta T cells. Isolated gammadelta T cells were found to undergo F-actin cytoskeleton reorganization when incubated with LTB(4) in vitro, confirming that gammadelta T lymphocytes can respond directly to LTB(4). In addition to its direct effect on gammadelta T cells, LTB(4) triggered their accumulation indirectly, via modulation of CCL2 production in mouse pleural cavities. These data show that gammadelta T cell migration into the pleural cavity of mice during diverse inflammatory responses is dependent on LTB(4)/BLT1.