Patricia Pacheco

Lipid Bodies Are Reservoirs of Cyclooxygenase-2 and Sites of Prostaglandin-E2 Synthesis in Colon Cancer Cells

Lipid bodies (lipid droplets) are emerging as dynamic organelles involved in lipid metabolism and inflammation. Increased lipid body numbers have been described in tumor cells; however, its functional significance in cancer has never been addressed. Here, we showed increased number of lipid bodies in tumor tissues from patients with adenocarcinoma of colon submitted to surgical resection when compared with an adjacent normal tissue. Accordingly, increased numbers of lipid bodies were observed in human colon adenocarcinoma cell lines and in a H-rasV12-transformed intestinal epithelial cell line (IEC-6 H-rasV12) compared with nontransformed IEC-6 cells. The functions of lipid bodies in eicosanoid synthesis in cancer cells were investigated. CACO-2 cells have increased expression of cyclooxygenase-2 (COX-2) when compared with IEC-6 cells. We showed by immunolocalization that, in addition to perinuclear stain, COX-2 and prostaglandin E (PGE) synthase present punctate cytoplasmic localizations that were concordant with adipose differentiation-related protein-labeled lipid bodies. The colocalization of COX-2 at lipid bodies was confirmed by immunoblot of subcellular fractionated cells. Direct localization of PGE(2) at its synthesis locale showed that lipid bodies are sources of eicosanoids in the transformed colon cancer cells. Treatment with either aspirin or the fatty acid synthase inhibitor C75 significantly reduced the number of lipid bodies and PGE(2) production in CACO-2 and in IEC-6 H-rasV12 cells with effects in cell proliferation. Together, our results showed that lipid bodies in colon cancer cells are dynamic and functional active organelles centrally involved in PGE(2) synthesis and may potentially have implications in the pathogenesis of adenocarcinoma of colon.

Lipopolysaccharide-Induced Leukocyte Lipid Body Formation In Vivo: Innate Immunity Elicited Intracellular Loci Involved in Eicosanoid Metabolism

Lipid bodies are rapidly inducible, specialized cytoplasmic domains for eicosanoid-forming enzyme localization, which we hypothesize to have specific roles in enhanced inflammatory mediator production during pathological conditions, including sepsis. However, little is known about the origins, composition, or functions of lipid bodies in vivo. We show that lipid body numbers were increased in leukocytes from septic patients in comparison with healthy subjects. Analogously, the intrathoracic administration of LPS into mice induced a dose- and time-dependent increase in lipid body numbers. Pretreatment with anti-CD14 or anti-CD11b/CD18 mAb drastically inhibited LPS-induced lipid body formation. Moreover, LPS failed to form lipid bodies in C3H/HeJ (TLR4 mutated) mice, demonstrating a requisite role for LPS receptors in lipid body formation. LPS-induced lipid body formation was also inhibited by the platelet-activating factor-receptor antagonists, suggesting a role for endogenous platelet-activating factor. The eicosanoid-forming enzymes, 5-lipoxygenase and cyclooxygenase-2, were immunolocalized within experimentally induced (LPS in mice) or naturally occurring (septic patients) lipid bodies. The proinflammatory cytokine involved in the pathogenesis of sepsis, TNF-α, was also shown to colocalize within lipid bodies. Prior stimulation of leukocytes to form lipid bodies enhanced the capacity of leukocytes to produce leukotriene B4 and PGE2. In conclusion, our studies indicate that lipid bodies formed after LPS stimulation and sepsis are sites for eicosanoid-forming enzymes and cytokine localization and may develop and function as structurally distinct, intracellular sites for paracrine eicosanoid synthesis during inflammatory conditions.

Contribution of macrophage migration inhibitory factor to the pathogenesis of dengue virus infection

Dengue fever is an emerging viral disease transmitted by arthropods to humans in tropical countries. Dengue hemorrhagic fever (DHF) is escalating in frequency and mortality rates. Here we studied the involvement of macrophage migration inhibitory factor (MIF) in dengue virus (DENV) infection and its pathogenesis. Patients with DHF had elevated plasma concentrations of MIF. Both leukocytes from these patients and macrophages from healthy donors infected in vitro with DENV showed a substantial amount of MIF within lipid droplets. The secretion of MIF by macrophages and hepatocytes required a productive infection and occurred without an increase in gene transcription or cell death, thus indicating active secretion from preformed stocks. In vivo infection of wild‐type and miFdeficient (Mif−/−) mice demonstrated a role of MIF in dengue pathogenesis. Clinical disease was less severe in Mif−/− mice, and they exhibited a significant delay in lethality, lower viremia, and lower viral load in the spleen than wild‐type mice. This reduction in all parameters of severity on DENV infection in Mif−/− mice correlated with reduced proinflam‐matory cytokine concentrations. These results demon‐strated the contribution of MIF to the pathogenesis of dengue and pointed to a possible beneficial role of neutralizing MIF as an adjunctive therapeutic approach to treat the severe forms of the disease.—Assuncäo‐Miranda, I., Amaral, F. A., Bozza, F. A., Fagundes, C. T., Sousa, L. P., Souza, D. G., Pacheco, P., Barbosa‐Lima, G., Gomes, R. N., Bozza, P. T., Da Poian, A. T., Teixeira, M. M., Bozza, M. T. Contribution of macro‐phage migration inhibitory factor to the pathogenesis of dengue virus infection. FASEB J. 24, 218–228 (2010). www.fasebj.org

Monocyte Chemoattractant Protein-1/CC Chemokine Ligand 2 Controls Microtubule-Driven Biogenesis and Leukotriene B4-Synthesizing Function of Macrophage Lipid Bodies Elicited by Innate Immune Response

Lipid bodies (also known as lipid droplets) are emerging as inflammatory organelles with roles in the innate immune response to infections and inflammatory processes. In this study, we identified MCP-1 as a key endogenous mediator of lipid body biogenesis in infection-driven inflammatory disorders and we described the cellular mechanisms and signaling pathways involved in the ability of MCP-1 to regulate the biogenesis and leukotriene B4 (LTB4) synthetic function of lipid bodies. In vivo assays in MCP-1−/− mice revealed that endogenous MCP-1 produced during polymicrobial infection or LPS-driven inflammatory responses has a critical role on the activation of lipid body-assembling machinery, as well as on empowering enzymatically these newly formed lipid bodies with LTB4 synthetic function within macrophages. MCP-1 triggered directly the rapid biogenesis of distinctive LTB4-synthesizing lipid bodies via CCR2-driven ERK- and PI3K-dependent intracellular signaling in in vitro-stimulated macrophages. Disturbance of microtubule organization by microtubule-active drugs demonstrated that MCP-1-induced lipid body biogenesis also signals through a pathway dependent on microtubular dynamics. Besides biogenic process, microtubules control LTB4-synthesizing function of MCP-1-elicited lipid bodies, in part by regulating the compartmentalization of key proteins, as adipose differentiation-related protein and 5-lipoxygenase. Therefore, infection-elicited MCP-1, besides its known CCR2-driven chemotactic function, appears as a key activator of lipid body biogenic and functional machineries, signaling through a microtubule-dependent manner.

MECHANISMS OF INCREASED SURVIVAL AFTER LIPOPOLYSACCHARIDE-INDUCED ENDOTOXIC SHOCK IN MICE CONSUMING OLIVE OIL-ENRICHED DIET

We examined the impact of dietary fatty acid intake on lipopolysaccharide (LPS)-induced endotoxic shock. C57Bl/6J mice were fed for 6 weeks with a commercial laboratory chow (CC) or with test chows containing 7% (w/w) canola oil (CO), sesame oil (SeO), soybean oil (SO), or virgin olive oil (OO). The increase in body weight and energy consumption were similar for all diets tested. In the sixth week, mice were injected intraperitoneally with 400 μg of bacterial LPS to induce endotoxic shock. LPS induced a massive neutrophil infiltration into the peritoneal cavity and an increase in lipid body (LB) formation in leukocytes recovered from the peritoneal fluid of mice fed with CC, CO, SeO, or SO. In addition, there were increases in prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and cytokines IL-6, IL-10, and MCP-1 in peritoneal lavage, as well as in plasma TNF-α. In contrast, mice fed with OO exhibited reduced neutrophil accumulation and LB formation, and also had lower levels of PGE2, LTB4, MCP-1, and TNF-α. All mice fed with CC, CO, SeO, or SO died within 48 to 72 h after LPS injection. Interestingly, mice fed with the OO diet were resistant to endotoxic shock, with 60% survival at 168 h. These data indicate that intake of OO may have a beneficial role, reducing the magnitude of the inflammatory process triggered by endotoxic shock through modulation of LB formation and of the production of inflammatory mediators.

Increased susceptibility to septic and endotoxic shock in monocyte chemoattractant protein 1/cc chemokine ligand 2-deficient mice correlates with reduced interleukin 10 and enhanced macrophage migration inhibitory factor production.

ABSTRACT The chemokine monocyte chemoattractant protein 1/CC chemokine ligand 2 (MCP-1/CCL2) is a potent chemoattractant of mononuclear cells and a regulatory mediator involved in a variety of inflammatory diseases. In the present study, we demonstrate that mcp-1/ccl2-deficient mice are more susceptible to systemic inflammatory response syndrome induced by lipopolysaccharide and to polymicrobial sepsis induced by cecum ligation and puncture (CLP) when compared with wild-type mice. Interestingly, in the CLP model, mcp-1/ccl2-deficient mice efficiently cleared the bacteria despite an impaired recruitment of leukocytes, especially mononuclear cells. The increased lethality rate in these models correlates with an impaired production of interleukin (IL) 10 in vivo. Furthermore, macrophages from mcp-1/ccl2-deficient mice activated with lipopolysaccharide also produced lower amounts of IL-10 and similar tumor necrosis factor compared with wild-type mice. We observed a drastic increase in the amounts of macrophage migration inhibitory factor at 6 and 24 h after CLP in mcp-1/ccl2-deficient mice. These results indicate that endogenous MCP-1/CCL2 positively regulates IL-10 but negatively controls macrophage migration inhibitory factor during peritoneal sepsis, thus suggesting an important immunomodulatory role for MCP-1/CCL2 in controlling the balance between proinflammatory and anti-inflammatory factors in sepsis.ABBREVIATIONS-CFU; colony forming unit, CLP; cecum ligation and puncture, H&E; hematoxilin/eosin, HMGB-1; high mobility group box, MCP-1/CCL2; monocyte chemoattractant protein-1/CC chemokine ligand-2, MIF; macrophage migration inhibitory factor, SIRS; systemic inflammatory response syndrome

Lipid bodies in oxidized LDL-induced foam cells are leukotriene-synthesizing organelles: a MCP-1/CCL2 regulated phenomenon

Lipid-laden foam macrophages are emerging as key players in early atherogenesis. Even though cytoplasmic lipid bodies (lipid droplets) are now recognized as organelles with cell functions beyond lipid storage, the mechanisms controlling lipid body biogenesis within macrophages and their additional functions in atherosclerosis are not completely elucidated. Here we studied oxLDL-elicited macrophage machinery involved in lipid body biogenesis as well as lipid body roles in leukotriene (LT) synthesis. Both in vivo and in vitro, oxLDL (but not native LDL) induced rapid assembly of cytoplasmic lipid bodies-bearing ADRP within mice macrophages. Such oxLDL-elicited foamy-like phenotype was a pertussis toxin-sensitive process that depended on a paracrine activity of endogenous MCP-1/CCL2 and activation of ERK. Pretreatment with neutralizing anti-MCP-1/CCL2 inhibited macrophage ADRP protein expression induced by oxLDL. By directly immuno-localizing leukotrienes at their sites of synthesis, we showed that oxLDL-induced newly formed lipid bodies function as active sites of LTB(4) and LTC(4) synthesis, since oxLDL-induced lipid bodies within foam macrophages compartmentalized the enzyme 5-lipoxygenase and five lipoxygenase-activating protein (FLAP) as well as newly formed LTB(4) and LTC(4). Consistent with MCP-1/CCL-2 role in ox-LDL-induced lipid body biogenesis, in CCR2 deficient mice both ox-LDL-induced lipid body assembly and LT release were reduced as compared to wild type mice. In conclusion, oxLDL-driven foam cells are enriched with leukotriene-synthesizing lipid bodies--specialized organelles whose biogenic process is mediated by MCP-1/CCL2-triggered CCR2 activation and ERK-dependent downstream signaling--that may amplify inflammatory mediator production in atherosclerosis.

Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development.

The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity.

Bradykinin down-regulates LPS-induced eosinophil accumulation in the pleural cavity of mice through type 2-kinin receptor activation: a role for prostaglandins

The role of both exogenously administered and endogenously generated bradykinin (BK) on LPS‐induced eosinophil accumulation in the mice pleural cavity was investigated by means of treatment with BK selective receptor agonists/antagonists and captopril. Intrathoracic (i.t.) injection of LPS (250 ng cavity−1) induced eosinophil influx at 24 h as previously described ( Bozza et al., 1993 ). Pretreatment with the B1 receptor antagonist des‐Arg9‐[leu‐8]BK (0.025 and 0.25 nmol cavity−1) showed no effect on this phenomenon, whereas pretreatment with the B2 receptor antagonists, NPC 17731 (0.025 and 0.25 nmol cavity−1) or HOE 140 (2.5 nmol cavity−1), increased LPS‐induced eosinophil influx. Accordingly, pretreatment with captopril at 10 mg kg−1 i.p., inhibited eosinophil infiltration induced by LPS in the pleural cavity, suggesting that endogenous BK is down‐regulating LPS‐induced eosinophil accumulation. BK administered at 15 and 25 nmol cavity−1, i.t. or i.p. also inhibited LPS‐induced eosinophil accumulation. BK alone had no effect on the basal number of leucocytes in the pleural or peritoneal cavity in doses up to 25 nmol cavity−1. Nevertheless, when injected at doses of 50 and 100 nmol cavity−1 BK induced leucocyte influx characterized by neutrophil and eosinophil accumulation at 24 h. Similarly to what was observed with BK, a specific B2 receptor agonist, Tyr8BK, administered at 0.25 nmol cavity−1 i.p., significantly inhibited the eosinophil influx induced by LPS. The mechanism by which B2 receptor agonists inhibit LPS‐induced eosinophil accumulation was investigated by pretreating the animals with indomethacin or a selective cyclooxygenase‐2 inhibitor, NS‐398. Pretreatment with either indomethacin or NS‐398 had no effect on eosinophil influx induced by LPS alone, but those drugs were able to restore the LPS‐induced eosinophil influx in Tyr8BK (0.25 nmol cavity−1) injected mice. In conclusion, endogenously generated bradykinin seems to modulate, through activation of B2 receptors, eosinophil accumulation induced by LPS via a mechanism dependent on prostanoid synthesis.

Effect of gedunin on acute articular inflammation and hypernociception in mice.

Gedunin, a natural limonoid from Meliaceae species, has been previously described as an antiinflammatory compound in experimental models of allergic inflammation. Here, we report the antiinflammatory and antinociceptive effects of gedunin in an acute model of articular inflammation induced by zymosan (500 μg/cavity; intra-articular) in C57BL/6 mice. Intraperitoneal (i.p.) pretreatment with gedunin (0.005–5 mg/kg) impaired zymosan-induced edema formation, neutrophil accumulation and hypernociception in mouse knee joints, due to decreased expression of preproET-1 mRNA and production of LTB4, PGE2, TNF-α and IL-6. Mouse post-treatment with gedunin (0.05 mg/kg; i.p.) 1 and 6 h after stimulation also impaired articular inflammation, by reverting edema formation, neutrophil accumulation and the production of lipid mediators, cytokines and endothelin. In addition, gedunin directly modulated the functions of neutrophils and macrophages in vitro. The pre-incubation of neutrophil with gedunin (100 µM) impaired shape change, adhesion to endothelial cells, chemotaxis and lipid body formation triggered by different stimuli. Macrophage pretreatment with gedunin impaired intracellular calcium mobilization, nitric oxide production, inducible nitric oxide synthase expression and induced the expression of the antiinflammatory chaperone heat shock protein 70. Our results demonstrate that gedunin presents remarkable antiinflammatory and anti-nociceptive effects on zymosan-induced inflamed knee joints, modulating different cell populations.