Cláudio Canetti - Academia.edu (original) (raw)

Papers by Cláudio Canetti

Research paper thumbnail of CCR4 Controls the Suppressive Effects of Regulatory T Cells on Early and Late Events during Severe Sepsis

PloS one, 2015

Sepsis is a deadly disease characterized by an overwhelming release of inflammatory mediators and... more Sepsis is a deadly disease characterized by an overwhelming release of inflammatory mediators and the activation of different types of cells. This altered state of cell activation, termed leukocyte reprogramming, contributes to patient outcome. However, the understanding of the process underlying sepsis and the role of regulatory T cells (Tregs) in sepsis remains to be elucidated. In this study, we investigated the role of CCR4, the CCL17/CCL22 chemokine receptor, in the innate and acquired immune responses during severe sepsis and the role of Tregs in effecting the outcome. In contrast with wild-type (WT) mice subjected to cecal ligation and puncture (CLP) sepsis, CCR4-deficient (CCR4-/-) septic mice presented an increased survival rate, significant neutrophil migration toward the infection site, a low bacterial count in the peritoneum, and reduced lung inflammation and serum cytokine levels. Thus, a better early host response may favor an adequate long-term response. Consequently,...

Research paper thumbnail of IL-18 enhances collagen-induced arthritis by recruiting neutrophils via TNF-alpha and leukotriene B4

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2003

IL-18 expression and functional activity have been associated with a range of autoimmune diseases... more IL-18 expression and functional activity have been associated with a range of autoimmune diseases. However, the precise mechanism by which IL-18 induces such pathology remains unclear. In this study we provide direct evidence that IL-18 activates neutrophils via TNF-alpha induction, which drives the production of leukotriene B(4) (LTB(4)), which in turn leads to neutrophil accumulation and subsequent local inflammation. rIL-18 administered i.p. resulted in the local synthesis of LTB(4) and a rapid influx of neutrophils into the peritoneal cavity, which could be effectively blocked by the LTB(4) synthesis inhibitor MK-886 (MK) or its receptor antagonist CP-105,696. IL-18-induced neutrophils recruitment and LTB(4) production could also be blocked by a neutralizing anti-TNF-alpha Ab. In addition, IL-18 failed to induce neutrophil accumulation in vivo in TNFRp55(-/-) mice. In an IL-18-dependent murine collagen-induced arthritis model, administration of MK significantly inhibited disease...

Research paper thumbnail of Age-Dependent Relevance of Endogenous 5-Lipoxygenase Derivatives in Anxiety-Like Behavior in Mice

PLoS ONE, 2014

When 5-lipoxygenase (5-LO) is inhibited, roughly half of the CNS effect of the prototypic endocan... more When 5-lipoxygenase (5-LO) is inhibited, roughly half of the CNS effect of the prototypic endocannabinoid anandamide (AEA) is lost. Therefore, we decided to investigate whether inhibiting this enzyme would influence physiological functions classically described as being under control of the endocannabinoid system. Although 5-LO inhibition by MK-886 reduced lipoxin A 4 levels in the brain, no effect was found in the elevated plus maze (EPM), even at the highest possible doses, via i.p. (10 mg/kg,) or i.c.v. (500 pmol/2 ml) routes. Accordingly, no alterations in anxiety-like behavior in the EPM test were observed in 5-LO KO mice. Interestingly, aged mice, which show reduced circulating lipoxin A 4 levels, were sensitive to MK-886, displaying an anxiogenic-like state in response to treatment. Moreover, exogenous lipoxin A 4 induced an anxiolytic-like profile in the EPM test. Our findings are in line with other reports showing no difference between FLAP KO or 5-LO KO and their control strains in adult mice, but increased anxiety-like behavior in aged mice. We also show for the first time that lipoxin A 4 affects mouse behavior. In conclusion, we propose an age-dependent relevancy of endogenous 5-LO derivatives in the modulation of anxiety-like behavior, in addition to a potential for exogenous lipoxin A 4 in producing an anxiolytic-like state.

Research paper thumbnail of 5-Lipoxygenase is a key determinant of acute myocardial inflammation and mortality during Trypanosoma cruzi infection

Microbes and Infection, 2010

This study provides evidence supporting the idea that although inflammatory cells migration to th... more 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 À/À mice exhibited reduced inflammation, collagen deposition, and migration of CD4 þ , CD8 þ , and IFN-g-producer cells compared with WT littermates. Moreover, decreased amounts of TNF-a, IFN-g, and nitric oxide synthase were found in the hearts of 5-LO À/À mice. Interestingly, despite of early higher parasitic load, 5-LO À/À 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 À/À mice, in which reduced myocarditis protects the animals during T. cruzi infection.

Research paper thumbnail of Proinflammatory Responses of Heme in Alveolar Macrophages: Repercussion in Lung Hemorrhagic Episodes

Mediators of Inflammation, 2013

Clinical and experimental observations have supported the notion that free heme released during h... more Clinical and experimental observations have supported the notion that free heme released during hemorrhagic and hemolytic episodes may have a major role in lung inflammation. With alveolar macrophages (AM) being the main line of defense in lung environments, the influence of free heme on AM activity and function was investigated. We observed that heme in a concentration range found during hemolytic episodes (3-30 M) elicits AM to present a proinflammatory profile, stimulating reactive oxygen species (ROS) and nitric oxide (NO) generation and inducing IL-1 , IL-6, and IL-10 secretion. ROS production is NADPH oxidasedependent, being inhibited by DPI and apocynin, and involves p47 subunit phosphorylation. Furthermore, heme induces NF-B nuclear translocation, iNOS, and also HO-1 expression. Moreover, AM stimulated with free heme show enhanced phagocytic and bactericidal activities. Taken together, the data support a dual role for heme in the inflammatory response associated with lung hemorrhage, acting as a proinflammatory molecule that can either act as both an adjuvant of the innate immunity and as an amplifier of the inflammatory response, leading tissue injury. The understanding of heme effects on pulmonary inflammatory processes can lead to the development of new strategies to ameliorate tissue damage associated with hemorrhagic episodes.

Research paper thumbnail of Leukotriene B4 mediates    T lymphocyte migration in response to diverse stimuli

Journal of Leukocyte Biology, 2010

Herein, we investigated the involvement of the 5-LOderived lipid mediator LTB 4 in ␥␦ T cell migr... more Herein, we investigated the involvement of the 5-LOderived lipid mediator LTB 4 in ␥␦ T cell migration. When injected into the i.pl. space of C57BL/6 mice, LTB 4 triggered ␥␦ 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 ␥␦ T cell accumulation into pleural cavities. Accordingly, 5-LO KO mice failed to recruit ␥␦ 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 ␥␦ 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 ␥␦ 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 ␥␦ T cells. Isolated ␥␦ T cells were found to undergo F-actin cytoskeleton reorganization when incubated with LTB 4 in vitro, confirming that ␥␦ T lymphocytes can respond directly to LTB 4 . In addition to its direct effect on ␥␦ T cells, LTB 4 triggered their accumulation indirectly, via modulation of CCL2 production in mouse pleural cavities. These data show that ␥␦ T cell migration into the pleural cavity of mice during diverse inflammatory responses is dependent on LTB 4 /BLT1. J. Leukoc. Biol. 87: 323-332; 2010.

Research paper thumbnail of Differential regulation by leukotrienes and calcium of Fc  receptor-induced phagocytosis and Syk activation in dendritic cells versus macrophages

Journal of Leukocyte Biology, 2006

or to block the high-affinity LTB 4 receptor BLT1. DCs were refractory to regulation by LTB 4 des... more or to block the high-affinity LTB 4 receptor BLT1. DCs were refractory to regulation by LTB 4 despite the fact that they expressed BLT1 and mobilized intracellular calcium in response to its ligation. This resistance to LTB 4 in DCs instead reflected the fact that in contrast to MØ, Syk activation in DCs was itself entirely independent of calcium. These results identify a fundamental difference in Fc␥R signaling between DCs and MØ, which may relate to the divergent, functional consequences of target ingestion in the two cell types.

Research paper thumbnail of Migration inhibitory factor (MIF) released by macrophages upon recognition of immune complexes is critical to inflammation in Arthus reaction

Journal of Leukocyte Biology, 2009

triggers Fc␥R-dependent inflammation, leading to tissue damage in rheumatoid arthritis, systemic ... more triggers Fc␥R-dependent inflammation, leading to tissue damage in rheumatoid arthritis, systemic lupus erythematous, immune glomerulonephritis, and several immune vasculitides. Evidences support a role for macrophage migration inhibitory factor (MIF) in a number of inflammatory diseases, but the triggering of its secretion and its physiopathological role upon IC deposition remain elusive. Herein, we show that human macrophages secreted MIF after IC recognition, which in turn controlled the secretion of TNF. Macrophages from Mif؊/؊ mice produced smaller amounts of TNF when stimulated with IgG-opsonized erythrocytes than wild-type (WT) cells. Using passive reverse Arthus reaction in the peritoneum and lungs as a model for IC-induced inflammation, we demonstrated that Mif؊/؊ mice had a milder response, observed by reduced neutrophil recruitment, vascular leakage, and secretion of TNF, MIP-2, and keratinocyte-derived chemokine compared with WT controls. Adoptive transfer of alveolar macrophages from WT to Mif؊/؊ mice rescued pulmonary neutrophil recruitment and TNF production upon passive reverse Arthus reaction. Our study indicates that Arthus inflammatory reaction is largely dependent on MIF and poses macrophages as a source of the MIF released upon IC recognition. These results give experimental support to the proposition that blockade of MIF might constitute an adjunctive, therapeutic approach to IC disease. J. Leukoc. Biol. 85: 855-861; 2009.

Research paper thumbnail of Critical Role of 5-Lipoxygenase and Heme Oxygenase-1 in Wound Healing

Journal of Investigative Dermatology, 2014

Lipid mediators derived from 5-lipoxygenase (5-LO) metabolism can activate both pro- and anti-inf... more 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.

Research paper thumbnail of Leukotriene B4 Modulates P2X7 Receptor-Mediated Leishmania amazonensis Elimination in Murine Macrophages

The Journal of Immunology, 2014

http://jimmunol.org/subscriptions is online at: The Journal of Immunology Information about subsc... more http://jimmunol.org/subscriptions is online at: The Journal of Immunology Information about subscribing to Permissions ATP is an important signaling molecule in the immune system, and it is able to bind the P2X7 purinergic receptor. Recently, our group showed that ATP-treated macrophages eliminate Leishmania amazonensis. It has been reported that leukotriene B 4 (LTB 4 ) reduces the parasitic load of infected macrophages. Additionally, it has been demonstrated that the P2X7 receptor can induce PLA 2 activation and arachidonic acid mobilization. Based on these findings, we investigated whether LTB 4 is produced upon P2X7 receptor activation and examined whether LTB 4 modulates parasite elimination. Using macrophages lacking the P2X7 receptor, we observed that ATP was not able to reduce L. amazonensis load. This result suggests a role of the P2X7 purinergic receptor in parasite elimination. In addition, ATP was sufficient to induce LTB 4 release from infected control macrophages but not from macrophages lacking the P2X7 receptor. Moreover, we found that ATP failed to decrease the parasitic load in 5-lipoxygenase (LO)-deficient macrophages. Treatment with the 5-LO inhibitor AA861 also impairs the ATP effect on parasitic loads. Furthermore, macrophages from 5-LO knockout mice eliminated L. amazonensis in the presence of exogenous LTB 4 , and macrophages obtained from P2X7 receptor knockout mice eliminated L. amazonensis when incubated with ionomycin. Finally, we demonstrated that in the presence of CP105696, an antagonist for LTB 4 high-affinity receptor, ATP was not able to reduce parasitic load. These results indicate that P2X7 receptor activation leads to LTB 4 formation, which is required for L. amazonensis elimination. FIGURE 8. Elimination of L. amazonensis via P2X7 receptor is mediated by LTB 4 . During infection with L. amazonensis, ATP is released into the extracellular medium. This ATP is then able to activate the P2X7 receptor, which causes activation of the LTB 4 production cascade. LTB 4 is also released and activates specific receptors such as BLT1 receptor. In a manner not yet known, BLT1 receptor activation leads to elimination of L. amazonensis in macrophages. VP La, L. amazonensis parasitophorous vacuole.

Research paper thumbnail of Activation of Phosphatase and Tensin Homolog on Chromosome 10 Mediates the Inhibition of Fc R Phagocytosis by Prostaglandin E2 in Alveolar Macrophages

The Journal of Immunology, 2007

PGE 2 has important inhibitory effects on the macrophage host defense functions of phagocytosis a... more PGE 2 has important inhibitory effects on the macrophage host defense functions of phagocytosis and killing, yet the molecular mechanisms involved remain to be fully elucidated. PGE 2 causes an elevation of cAMP in alveolar macrophages (AMs), which in turn activates the cAMP effector targets, protein kinase A and the exchange protein activated by cAMP (Epac)-1. We now report that Fc␥R-induced PI3K/Akt and ERK-1/2 activation are inhibited by PGE 2 in AMs. By specifically inhibiting the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in AMs, we attenuated the inhibitory effects of both PGE 2 and a specific Epac-1 agonist (8-pCPT-2-O-Me-cAMP) on Fc␥R-mediated phagocytosis and Akt/ERK-1/2 activation; PTEN inhibition also decreased PGE 2 -induced suppression of bacterial killing by AMs. Moreover, PGE 2 and the Epac-1 agonist induced an increase in PTEN lipid phosphatase activity, and this was associated with decreased tyrosine phosphorylation on PTEN-a mechanism known to regulate PTEN activity. Using a pharmacological approach, we demonstrated a role for Src homology 2-containing protein tyrosine phosphatase-1 in the PGE 2 -induced tyrosine dephosphorylation of PTEN. Collectively, these data reveal that PGE 2 , via Epac-1 activation, enhances SHP-1 activity, resulting in increased PTEN activity. We suggest that this mechanism contributes to the ability of PGE 2 to inhibit PI3K-dependent innate immune signaling in primary macrophages. Abbreviations used in this paper: PIP3, phosphatidylinositol 3,4,5-triphosphate; PTEN, phosphatase and tensin homolog deleted on chromosome 10; AM, alveolar macrophage; Epac-1, exchange protein activated by cAMP-1; PKA, protein kinase A; SHP-1, Src homology 2-containing protein tyrosine phosphatase-1; SHPI, ␣-bromo-4-(carboxymethoxy)-acetophenone.

Research paper thumbnail of Tumour necrosis factor-α mediates neutrophil migration to the knee synovial cavity during immune inflammation

European Journal of Pharmacology, 2004

Tumour necrosis factor (TNF)-alpha, interleukin-1beta, interleukin-8 and leukotriene B4 have an i... more Tumour necrosis factor (TNF)-alpha, interleukin-1beta, interleukin-8 and leukotriene B4 have an important role on neutrophil recruitment during immune-inflammation. Here we evaluated the participation of several inflammatory mediators on ovalbumin-induced neutrophil recruitment in the knee articular space of immunized rats. Ovalbumin administration in immunized, but not in control, rats induced a dose- and time-dependent neutrophil accumulation, which was inhibited by dexamethasone, pentoxifylline or thalidomide, but not by selective inhibitors of nitric oxide (nitro-L-arginine), platelet-activating factor (BN50730 or UK74505), prostaglandins (indomethacin), histamine (meclisine) or leukotriene B4 (MK 886 and CP105,696). Anti-TNF-alpha antiserum, but not anti-interleukin-1beta or anti-CINC-1 (cytokine-induced neutrophil chemoattractant 1) antisera, impaired ovalbumin-induced neutrophil accumulation. High amounts of TNF-alpha were detected in the exudates, which was inhibited by dexamethasone, pentoxifylline and thalidomide. These results suggest a specific role for TNF-alpha in this model, and the ability of pentoxifylline and thalidomide to inhibit both neutrophil influx and TNF-alpha release may have therapeutic implications in arthritis.

Research paper thumbnail of Syk activation is a leukotriene B4-regulated event involved in macrophage phagocytosis of IgG-coated targets but not apoptotic cells

Blood, 2003

Macrophages are called upon to ingest both IgG-coated targets and apoptotic cells. Important role... more Macrophages are called upon to ingest both IgG-coated targets and apoptotic cells. Important roles for tyrosine kinase Syk and leukotriene B4 (LTB4) are recognized in FcgammaR-mediated phagocytosis. Here we evaluated the roles of Syk and LTB4 in macrophage phagocytosis of apoptotic thymocytes versus IgG-coated erythrocytes. Macrophage ingestion of apoptotic thymocytes was not influenced by exogenous or endogenous LTB4 nor associated with Syk activation (phosphorylation). By contrast, LTB4 dose-dependently amplified FcgammaR-mediated phagocytosis as well as Syk activation. Furthermore, a role for endogenous LTB4 in Syk activation during FcgammaR-mediated phagocytosis was demonstrated using pharmacologic and genetic abrogation of 5-lipoxygenase. LTB4 was unique among 5-lipoxygenase products in this regard, since LTD4 and 5-hydroxyeicosatetraenoic acid (HETE) were unable to amplify Syk activation in response to FcgammaR engagement. Ca2+ chelation studies revealed that FcgammaR-mediated Syk activation as well as LTB4 amplification thereof was Ca2+ regulated. These 2 parallel phagocytic processes therefore exhibit initial divergence in signal transduction events, with Syk activation being an LTB4-regulated event in FcgammaR-mediated but not apoptotic cell ingestion. As LTB4 is an important proinflammatory product of macrophages, we speculate that this divergence evolved to permit FcgammaR-mediated phagocytosis to proceed in an inflammatory milieu, while apoptotic cell clearance is noninflammatory.

Research paper thumbnail of Leukotriene B4 mediates vascular smooth muscle cell migration through αvβ3 integrin transactivation

Atherosclerosis, 2010

Vascular injury leads to a local inflammatory response, characterized by endothelial damage, extr... more Vascular injury leads to a local inflammatory response, characterized by endothelial damage, extracellular matrix exposition and aggregation/adhesion of platelets and circulating leukocytes. The release of inflammatory mediators amplifies the process, and can induce vascular smooth muscle cells (SMC) migration and proliferation. Released by leukocytes, leukotriene B4 (LTB4) induces reactive oxygen species production and SMC chemotaxis. This study was conducted to elucidate the molecular mechanisms involved in the effect of LTB4 on SMC migration, and a rat linage of vascular SMC (A7r5) were used throughout. The chemotactic effect of LTB4 was dependent on the concentration used, being comparable to AngII at 100 nM. Migration induced by LTB4 was inhibited in the presence of pertussis toxin, CP-105696, a BLT1 receptor antagonist, and by LY294002 or PD98059, two inhibitors of PI3K and MEK1/2, respectively. Stimulation of SMC with LTB4 triggered integrin-associated signaling pathways, inducing focal adhesion kinase (FAK) phosphorylation, mobilization of actin cytoskeleton, association of FAK to PI3K, ERK-2 phosphorylation and nuclear translocation, and also NFκB pathway activation. Pretreatment of SMC with a selective ligand of αvβ3 integrin, kistrin, inhibited LTB4-induced chemotaxis, FAK phosphorylation, FAK-PI3K association, and also inhibited ERK-2 and NFκB pathways activation. Taken together, the data demonstrated, for the first time, that the effect of LTB4 on SMC migration is modulated by integrin signaling activation, suggesting that these adhesion molecules might be important target for therapeutic intervention in cardiovascular diseases.

Research paper thumbnail of 5-Lypoxygenase Products Are Involved in Renal Tubulointerstitial Injury Induced by Albumin Overload in Proximal Tubules in Mice

PLoS ONE, 2014

The role of albumin overload in proximal tubules (PT) in the development of tubulointerstitial in... more The role of albumin overload in proximal tubules (PT) in the development of tubulointerstitial injury and, consequently, in the progression of renal disease has become more relevant in recent years. Despite the importance of leukotrienes (LTs) in renal disease, little is known about their role in tubulointerstitial injury. The aim of the present work was to investigate the possible role of LTs on tubulointerstitial injury induced by albumin overload. An animal model of tubulointerstitial injury challenged by bovine serum albumin was developed in SV129 mice (wild-type) and 5-lipoxygenase-deficient mice (5-LO -/-). The changes in glomerular morphology and nestin expression observed in wild-type mice subjected to kidney insult were also observed in 5-LO -/mice. The levels of urinary protein observed in the 5-LO -/mice subjected or not to kidney insult were lower than those observed in respective wild-type mice. Furthermore, the increase in lactate dehydrogenase activity, a marker of tubule damage, observed in wild-type mice subjected to kidney insult did not occur in 5-LO -/mice. LTB 4 and LTD 4 , 5-LO products, decreased the uptake of albumin in LLC-PK1 cells, a well-characterized porcine PT cell line. This effect correlated with activation of protein kinase C and inhibition of protein kinase B. The level of proinflammatory cytokines, tumor necrosis factor-a and interleukin (IL)-6, increased in mice subjected to kidney insult but this effect was not modified in 5-LO -/mice. However, 5-LO -/mice subjected to kidney insult presented lower macrophage infiltration and higher levels of IL-10 than wild-type mice. Our results reveal that LTs have an important role in tubulointerstitial disease induced by albumin overload.

Research paper thumbnail of CCR4 Controls the Suppressive Effects of Regulatory T Cells on Early and Late Events during Severe Sepsis

PloS one, 2015

Sepsis is a deadly disease characterized by an overwhelming release of inflammatory mediators and... more Sepsis is a deadly disease characterized by an overwhelming release of inflammatory mediators and the activation of different types of cells. This altered state of cell activation, termed leukocyte reprogramming, contributes to patient outcome. However, the understanding of the process underlying sepsis and the role of regulatory T cells (Tregs) in sepsis remains to be elucidated. In this study, we investigated the role of CCR4, the CCL17/CCL22 chemokine receptor, in the innate and acquired immune responses during severe sepsis and the role of Tregs in effecting the outcome. In contrast with wild-type (WT) mice subjected to cecal ligation and puncture (CLP) sepsis, CCR4-deficient (CCR4-/-) septic mice presented an increased survival rate, significant neutrophil migration toward the infection site, a low bacterial count in the peritoneum, and reduced lung inflammation and serum cytokine levels. Thus, a better early host response may favor an adequate long-term response. Consequently,...

Research paper thumbnail of IL-18 enhances collagen-induced arthritis by recruiting neutrophils via TNF-alpha and leukotriene B4

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2003

IL-18 expression and functional activity have been associated with a range of autoimmune diseases... more IL-18 expression and functional activity have been associated with a range of autoimmune diseases. However, the precise mechanism by which IL-18 induces such pathology remains unclear. In this study we provide direct evidence that IL-18 activates neutrophils via TNF-alpha induction, which drives the production of leukotriene B(4) (LTB(4)), which in turn leads to neutrophil accumulation and subsequent local inflammation. rIL-18 administered i.p. resulted in the local synthesis of LTB(4) and a rapid influx of neutrophils into the peritoneal cavity, which could be effectively blocked by the LTB(4) synthesis inhibitor MK-886 (MK) or its receptor antagonist CP-105,696. IL-18-induced neutrophils recruitment and LTB(4) production could also be blocked by a neutralizing anti-TNF-alpha Ab. In addition, IL-18 failed to induce neutrophil accumulation in vivo in TNFRp55(-/-) mice. In an IL-18-dependent murine collagen-induced arthritis model, administration of MK significantly inhibited disease...

Research paper thumbnail of Age-Dependent Relevance of Endogenous 5-Lipoxygenase Derivatives in Anxiety-Like Behavior in Mice

PLoS ONE, 2014

When 5-lipoxygenase (5-LO) is inhibited, roughly half of the CNS effect of the prototypic endocan... more When 5-lipoxygenase (5-LO) is inhibited, roughly half of the CNS effect of the prototypic endocannabinoid anandamide (AEA) is lost. Therefore, we decided to investigate whether inhibiting this enzyme would influence physiological functions classically described as being under control of the endocannabinoid system. Although 5-LO inhibition by MK-886 reduced lipoxin A 4 levels in the brain, no effect was found in the elevated plus maze (EPM), even at the highest possible doses, via i.p. (10 mg/kg,) or i.c.v. (500 pmol/2 ml) routes. Accordingly, no alterations in anxiety-like behavior in the EPM test were observed in 5-LO KO mice. Interestingly, aged mice, which show reduced circulating lipoxin A 4 levels, were sensitive to MK-886, displaying an anxiogenic-like state in response to treatment. Moreover, exogenous lipoxin A 4 induced an anxiolytic-like profile in the EPM test. Our findings are in line with other reports showing no difference between FLAP KO or 5-LO KO and their control strains in adult mice, but increased anxiety-like behavior in aged mice. We also show for the first time that lipoxin A 4 affects mouse behavior. In conclusion, we propose an age-dependent relevancy of endogenous 5-LO derivatives in the modulation of anxiety-like behavior, in addition to a potential for exogenous lipoxin A 4 in producing an anxiolytic-like state.

Research paper thumbnail of 5-Lipoxygenase is a key determinant of acute myocardial inflammation and mortality during Trypanosoma cruzi infection

Microbes and Infection, 2010

This study provides evidence supporting the idea that although inflammatory cells migration to th... more 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 À/À mice exhibited reduced inflammation, collagen deposition, and migration of CD4 þ , CD8 þ , and IFN-g-producer cells compared with WT littermates. Moreover, decreased amounts of TNF-a, IFN-g, and nitric oxide synthase were found in the hearts of 5-LO À/À mice. Interestingly, despite of early higher parasitic load, 5-LO À/À 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 À/À mice, in which reduced myocarditis protects the animals during T. cruzi infection.

Research paper thumbnail of Proinflammatory Responses of Heme in Alveolar Macrophages: Repercussion in Lung Hemorrhagic Episodes

Mediators of Inflammation, 2013

Clinical and experimental observations have supported the notion that free heme released during h... more Clinical and experimental observations have supported the notion that free heme released during hemorrhagic and hemolytic episodes may have a major role in lung inflammation. With alveolar macrophages (AM) being the main line of defense in lung environments, the influence of free heme on AM activity and function was investigated. We observed that heme in a concentration range found during hemolytic episodes (3-30 M) elicits AM to present a proinflammatory profile, stimulating reactive oxygen species (ROS) and nitric oxide (NO) generation and inducing IL-1 , IL-6, and IL-10 secretion. ROS production is NADPH oxidasedependent, being inhibited by DPI and apocynin, and involves p47 subunit phosphorylation. Furthermore, heme induces NF-B nuclear translocation, iNOS, and also HO-1 expression. Moreover, AM stimulated with free heme show enhanced phagocytic and bactericidal activities. Taken together, the data support a dual role for heme in the inflammatory response associated with lung hemorrhage, acting as a proinflammatory molecule that can either act as both an adjuvant of the innate immunity and as an amplifier of the inflammatory response, leading tissue injury. The understanding of heme effects on pulmonary inflammatory processes can lead to the development of new strategies to ameliorate tissue damage associated with hemorrhagic episodes.

Research paper thumbnail of Leukotriene B4 mediates    T lymphocyte migration in response to diverse stimuli

Journal of Leukocyte Biology, 2010

Herein, we investigated the involvement of the 5-LOderived lipid mediator LTB 4 in ␥␦ T cell migr... more Herein, we investigated the involvement of the 5-LOderived lipid mediator LTB 4 in ␥␦ T cell migration. When injected into the i.pl. space of C57BL/6 mice, LTB 4 triggered ␥␦ 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 ␥␦ T cell accumulation into pleural cavities. Accordingly, 5-LO KO mice failed to recruit ␥␦ 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 ␥␦ 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 ␥␦ 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 ␥␦ T cells. Isolated ␥␦ T cells were found to undergo F-actin cytoskeleton reorganization when incubated with LTB 4 in vitro, confirming that ␥␦ T lymphocytes can respond directly to LTB 4 . In addition to its direct effect on ␥␦ T cells, LTB 4 triggered their accumulation indirectly, via modulation of CCL2 production in mouse pleural cavities. These data show that ␥␦ T cell migration into the pleural cavity of mice during diverse inflammatory responses is dependent on LTB 4 /BLT1. J. Leukoc. Biol. 87: 323-332; 2010.

Research paper thumbnail of Differential regulation by leukotrienes and calcium of Fc  receptor-induced phagocytosis and Syk activation in dendritic cells versus macrophages

Journal of Leukocyte Biology, 2006

or to block the high-affinity LTB 4 receptor BLT1. DCs were refractory to regulation by LTB 4 des... more or to block the high-affinity LTB 4 receptor BLT1. DCs were refractory to regulation by LTB 4 despite the fact that they expressed BLT1 and mobilized intracellular calcium in response to its ligation. This resistance to LTB 4 in DCs instead reflected the fact that in contrast to MØ, Syk activation in DCs was itself entirely independent of calcium. These results identify a fundamental difference in Fc␥R signaling between DCs and MØ, which may relate to the divergent, functional consequences of target ingestion in the two cell types.

Research paper thumbnail of Migration inhibitory factor (MIF) released by macrophages upon recognition of immune complexes is critical to inflammation in Arthus reaction

Journal of Leukocyte Biology, 2009

triggers Fc␥R-dependent inflammation, leading to tissue damage in rheumatoid arthritis, systemic ... more triggers Fc␥R-dependent inflammation, leading to tissue damage in rheumatoid arthritis, systemic lupus erythematous, immune glomerulonephritis, and several immune vasculitides. Evidences support a role for macrophage migration inhibitory factor (MIF) in a number of inflammatory diseases, but the triggering of its secretion and its physiopathological role upon IC deposition remain elusive. Herein, we show that human macrophages secreted MIF after IC recognition, which in turn controlled the secretion of TNF. Macrophages from Mif؊/؊ mice produced smaller amounts of TNF when stimulated with IgG-opsonized erythrocytes than wild-type (WT) cells. Using passive reverse Arthus reaction in the peritoneum and lungs as a model for IC-induced inflammation, we demonstrated that Mif؊/؊ mice had a milder response, observed by reduced neutrophil recruitment, vascular leakage, and secretion of TNF, MIP-2, and keratinocyte-derived chemokine compared with WT controls. Adoptive transfer of alveolar macrophages from WT to Mif؊/؊ mice rescued pulmonary neutrophil recruitment and TNF production upon passive reverse Arthus reaction. Our study indicates that Arthus inflammatory reaction is largely dependent on MIF and poses macrophages as a source of the MIF released upon IC recognition. These results give experimental support to the proposition that blockade of MIF might constitute an adjunctive, therapeutic approach to IC disease. J. Leukoc. Biol. 85: 855-861; 2009.

Research paper thumbnail of Critical Role of 5-Lipoxygenase and Heme Oxygenase-1 in Wound Healing

Journal of Investigative Dermatology, 2014

Lipid mediators derived from 5-lipoxygenase (5-LO) metabolism can activate both pro- and anti-inf... more 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.

Research paper thumbnail of Leukotriene B4 Modulates P2X7 Receptor-Mediated Leishmania amazonensis Elimination in Murine Macrophages

The Journal of Immunology, 2014

http://jimmunol.org/subscriptions is online at: The Journal of Immunology Information about subsc... more http://jimmunol.org/subscriptions is online at: The Journal of Immunology Information about subscribing to Permissions ATP is an important signaling molecule in the immune system, and it is able to bind the P2X7 purinergic receptor. Recently, our group showed that ATP-treated macrophages eliminate Leishmania amazonensis. It has been reported that leukotriene B 4 (LTB 4 ) reduces the parasitic load of infected macrophages. Additionally, it has been demonstrated that the P2X7 receptor can induce PLA 2 activation and arachidonic acid mobilization. Based on these findings, we investigated whether LTB 4 is produced upon P2X7 receptor activation and examined whether LTB 4 modulates parasite elimination. Using macrophages lacking the P2X7 receptor, we observed that ATP was not able to reduce L. amazonensis load. This result suggests a role of the P2X7 purinergic receptor in parasite elimination. In addition, ATP was sufficient to induce LTB 4 release from infected control macrophages but not from macrophages lacking the P2X7 receptor. Moreover, we found that ATP failed to decrease the parasitic load in 5-lipoxygenase (LO)-deficient macrophages. Treatment with the 5-LO inhibitor AA861 also impairs the ATP effect on parasitic loads. Furthermore, macrophages from 5-LO knockout mice eliminated L. amazonensis in the presence of exogenous LTB 4 , and macrophages obtained from P2X7 receptor knockout mice eliminated L. amazonensis when incubated with ionomycin. Finally, we demonstrated that in the presence of CP105696, an antagonist for LTB 4 high-affinity receptor, ATP was not able to reduce parasitic load. These results indicate that P2X7 receptor activation leads to LTB 4 formation, which is required for L. amazonensis elimination. FIGURE 8. Elimination of L. amazonensis via P2X7 receptor is mediated by LTB 4 . During infection with L. amazonensis, ATP is released into the extracellular medium. This ATP is then able to activate the P2X7 receptor, which causes activation of the LTB 4 production cascade. LTB 4 is also released and activates specific receptors such as BLT1 receptor. In a manner not yet known, BLT1 receptor activation leads to elimination of L. amazonensis in macrophages. VP La, L. amazonensis parasitophorous vacuole.

Research paper thumbnail of Activation of Phosphatase and Tensin Homolog on Chromosome 10 Mediates the Inhibition of Fc R Phagocytosis by Prostaglandin E2 in Alveolar Macrophages

The Journal of Immunology, 2007

PGE 2 has important inhibitory effects on the macrophage host defense functions of phagocytosis a... more PGE 2 has important inhibitory effects on the macrophage host defense functions of phagocytosis and killing, yet the molecular mechanisms involved remain to be fully elucidated. PGE 2 causes an elevation of cAMP in alveolar macrophages (AMs), which in turn activates the cAMP effector targets, protein kinase A and the exchange protein activated by cAMP (Epac)-1. We now report that Fc␥R-induced PI3K/Akt and ERK-1/2 activation are inhibited by PGE 2 in AMs. By specifically inhibiting the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in AMs, we attenuated the inhibitory effects of both PGE 2 and a specific Epac-1 agonist (8-pCPT-2-O-Me-cAMP) on Fc␥R-mediated phagocytosis and Akt/ERK-1/2 activation; PTEN inhibition also decreased PGE 2 -induced suppression of bacterial killing by AMs. Moreover, PGE 2 and the Epac-1 agonist induced an increase in PTEN lipid phosphatase activity, and this was associated with decreased tyrosine phosphorylation on PTEN-a mechanism known to regulate PTEN activity. Using a pharmacological approach, we demonstrated a role for Src homology 2-containing protein tyrosine phosphatase-1 in the PGE 2 -induced tyrosine dephosphorylation of PTEN. Collectively, these data reveal that PGE 2 , via Epac-1 activation, enhances SHP-1 activity, resulting in increased PTEN activity. We suggest that this mechanism contributes to the ability of PGE 2 to inhibit PI3K-dependent innate immune signaling in primary macrophages. Abbreviations used in this paper: PIP3, phosphatidylinositol 3,4,5-triphosphate; PTEN, phosphatase and tensin homolog deleted on chromosome 10; AM, alveolar macrophage; Epac-1, exchange protein activated by cAMP-1; PKA, protein kinase A; SHP-1, Src homology 2-containing protein tyrosine phosphatase-1; SHPI, ␣-bromo-4-(carboxymethoxy)-acetophenone.

Research paper thumbnail of Tumour necrosis factor-α mediates neutrophil migration to the knee synovial cavity during immune inflammation

European Journal of Pharmacology, 2004

Tumour necrosis factor (TNF)-alpha, interleukin-1beta, interleukin-8 and leukotriene B4 have an i... more Tumour necrosis factor (TNF)-alpha, interleukin-1beta, interleukin-8 and leukotriene B4 have an important role on neutrophil recruitment during immune-inflammation. Here we evaluated the participation of several inflammatory mediators on ovalbumin-induced neutrophil recruitment in the knee articular space of immunized rats. Ovalbumin administration in immunized, but not in control, rats induced a dose- and time-dependent neutrophil accumulation, which was inhibited by dexamethasone, pentoxifylline or thalidomide, but not by selective inhibitors of nitric oxide (nitro-L-arginine), platelet-activating factor (BN50730 or UK74505), prostaglandins (indomethacin), histamine (meclisine) or leukotriene B4 (MK 886 and CP105,696). Anti-TNF-alpha antiserum, but not anti-interleukin-1beta or anti-CINC-1 (cytokine-induced neutrophil chemoattractant 1) antisera, impaired ovalbumin-induced neutrophil accumulation. High amounts of TNF-alpha were detected in the exudates, which was inhibited by dexamethasone, pentoxifylline and thalidomide. These results suggest a specific role for TNF-alpha in this model, and the ability of pentoxifylline and thalidomide to inhibit both neutrophil influx and TNF-alpha release may have therapeutic implications in arthritis.

Research paper thumbnail of Syk activation is a leukotriene B4-regulated event involved in macrophage phagocytosis of IgG-coated targets but not apoptotic cells

Blood, 2003

Macrophages are called upon to ingest both IgG-coated targets and apoptotic cells. Important role... more Macrophages are called upon to ingest both IgG-coated targets and apoptotic cells. Important roles for tyrosine kinase Syk and leukotriene B4 (LTB4) are recognized in FcgammaR-mediated phagocytosis. Here we evaluated the roles of Syk and LTB4 in macrophage phagocytosis of apoptotic thymocytes versus IgG-coated erythrocytes. Macrophage ingestion of apoptotic thymocytes was not influenced by exogenous or endogenous LTB4 nor associated with Syk activation (phosphorylation). By contrast, LTB4 dose-dependently amplified FcgammaR-mediated phagocytosis as well as Syk activation. Furthermore, a role for endogenous LTB4 in Syk activation during FcgammaR-mediated phagocytosis was demonstrated using pharmacologic and genetic abrogation of 5-lipoxygenase. LTB4 was unique among 5-lipoxygenase products in this regard, since LTD4 and 5-hydroxyeicosatetraenoic acid (HETE) were unable to amplify Syk activation in response to FcgammaR engagement. Ca2+ chelation studies revealed that FcgammaR-mediated Syk activation as well as LTB4 amplification thereof was Ca2+ regulated. These 2 parallel phagocytic processes therefore exhibit initial divergence in signal transduction events, with Syk activation being an LTB4-regulated event in FcgammaR-mediated but not apoptotic cell ingestion. As LTB4 is an important proinflammatory product of macrophages, we speculate that this divergence evolved to permit FcgammaR-mediated phagocytosis to proceed in an inflammatory milieu, while apoptotic cell clearance is noninflammatory.

Research paper thumbnail of Leukotriene B4 mediates vascular smooth muscle cell migration through αvβ3 integrin transactivation

Atherosclerosis, 2010

Vascular injury leads to a local inflammatory response, characterized by endothelial damage, extr... more Vascular injury leads to a local inflammatory response, characterized by endothelial damage, extracellular matrix exposition and aggregation/adhesion of platelets and circulating leukocytes. The release of inflammatory mediators amplifies the process, and can induce vascular smooth muscle cells (SMC) migration and proliferation. Released by leukocytes, leukotriene B4 (LTB4) induces reactive oxygen species production and SMC chemotaxis. This study was conducted to elucidate the molecular mechanisms involved in the effect of LTB4 on SMC migration, and a rat linage of vascular SMC (A7r5) were used throughout. The chemotactic effect of LTB4 was dependent on the concentration used, being comparable to AngII at 100 nM. Migration induced by LTB4 was inhibited in the presence of pertussis toxin, CP-105696, a BLT1 receptor antagonist, and by LY294002 or PD98059, two inhibitors of PI3K and MEK1/2, respectively. Stimulation of SMC with LTB4 triggered integrin-associated signaling pathways, inducing focal adhesion kinase (FAK) phosphorylation, mobilization of actin cytoskeleton, association of FAK to PI3K, ERK-2 phosphorylation and nuclear translocation, and also NFκB pathway activation. Pretreatment of SMC with a selective ligand of αvβ3 integrin, kistrin, inhibited LTB4-induced chemotaxis, FAK phosphorylation, FAK-PI3K association, and also inhibited ERK-2 and NFκB pathways activation. Taken together, the data demonstrated, for the first time, that the effect of LTB4 on SMC migration is modulated by integrin signaling activation, suggesting that these adhesion molecules might be important target for therapeutic intervention in cardiovascular diseases.

Research paper thumbnail of 5-Lypoxygenase Products Are Involved in Renal Tubulointerstitial Injury Induced by Albumin Overload in Proximal Tubules in Mice

PLoS ONE, 2014

The role of albumin overload in proximal tubules (PT) in the development of tubulointerstitial in... more The role of albumin overload in proximal tubules (PT) in the development of tubulointerstitial injury and, consequently, in the progression of renal disease has become more relevant in recent years. Despite the importance of leukotrienes (LTs) in renal disease, little is known about their role in tubulointerstitial injury. The aim of the present work was to investigate the possible role of LTs on tubulointerstitial injury induced by albumin overload. An animal model of tubulointerstitial injury challenged by bovine serum albumin was developed in SV129 mice (wild-type) and 5-lipoxygenase-deficient mice (5-LO -/-). The changes in glomerular morphology and nestin expression observed in wild-type mice subjected to kidney insult were also observed in 5-LO -/mice. The levels of urinary protein observed in the 5-LO -/mice subjected or not to kidney insult were lower than those observed in respective wild-type mice. Furthermore, the increase in lactate dehydrogenase activity, a marker of tubule damage, observed in wild-type mice subjected to kidney insult did not occur in 5-LO -/mice. LTB 4 and LTD 4 , 5-LO products, decreased the uptake of albumin in LLC-PK1 cells, a well-characterized porcine PT cell line. This effect correlated with activation of protein kinase C and inhibition of protein kinase B. The level of proinflammatory cytokines, tumor necrosis factor-a and interleukin (IL)-6, increased in mice subjected to kidney insult but this effect was not modified in 5-LO -/mice. However, 5-LO -/mice subjected to kidney insult presented lower macrophage infiltration and higher levels of IL-10 than wild-type mice. Our results reveal that LTs have an important role in tubulointerstitial disease induced by albumin overload.