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Prostaglandins D2 and E2 have opposite effects on alveolar macrophages infected with Histoplasma capsulatum
2017; Elsevier BV; Volume: 59; Issue: 2 Linguagem: Inglês
10.1194/jlr.m078162
ISSN1539-7262
AutoresPriscilla Aparecida Tártari Pereira, Patrícia A. Assis, Morgana Kelly Borges Prado, Simone G. Ramos, David M. Aronoff, Francisco Wanderley Garcia Paula‐Silva, Carlos A. Sorgi, Lúcia Helena Faccioli,
Tópico(s)Toxoplasma gondii Research Studies
ResumoProstaglandin E2 (PGE2) suppresses macrophage effector mechanisms; however, little is known about the function of PGD2 in infected alveolar macrophages (AMs). Using serum-opsonized Histoplasma capsulatum (Ops-H. capsulatum) in vitro, we demonstrated that AMs produced PGE2 and PGD2 in a time-dependent manner, with PGE2 levels exceeding those of PGD2 by 48 h postinfection. Comparison of the effects of both exogenous PGs on AMs revealed that PGD2 increased phagocytosis and killing through the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes receptor, whereas PGE2 had opposite effects, through E prostanoid (EP) receptor 2 (EP2)/EP4-dependent mechanisms. Moreover, PGD2 inhibited phospholipase C-γ (PLC-γ) phosphorylation, reduced IL-10 production, and increased leukotriene B4 receptor expression. In contrast, exogenous PGE2 treatment reduced PLC-γ phosphorylation, p38 and nuclear factor κB activation, TNF-α, H2O2, and leukotriene B4, but increased IL-1β production. Using specific compounds to inhibit the synthesis of each PG in vitro and in vivo, we found that endogenous PGD2 contributed to fungicidal mechanisms and controlled inflammation, whereas endogenous PGE2 decreased phagocytosis and killing of the fungus and induced inflammation. These findings demonstrate that, although PGD2 acts as an immunostimulatory mediator to control H. capsulatum infection, PGE2 has immunosuppressive effects, and the balance between these two PGs may limit collateral immune damage at the expense of microbial containment. Prostaglandin E2 (PGE2) suppresses macrophage effector mechanisms; however, little is known about the function of PGD2 in infected alveolar macrophages (AMs). Using serum-opsonized Histoplasma capsulatum (Ops-H. capsulatum) in vitro, we demonstrated that AMs produced PGE2 and PGD2 in a time-dependent manner, with PGE2 levels exceeding those of PGD2 by 48 h postinfection. Comparison of the effects of both exogenous PGs on AMs revealed that PGD2 increased phagocytosis and killing through the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes receptor, whereas PGE2 had opposite effects, through E prostanoid (EP) receptor 2 (EP2)/EP4-dependent mechanisms. Moreover, PGD2 inhibited phospholipase C-γ (PLC-γ) phosphorylation, reduced IL-10 production, and increased leukotriene B4 receptor expression. In contrast, exogenous PGE2 treatment reduced PLC-γ phosphorylation, p38 and nuclear factor κB activation, TNF-α, H2O2, and leukotriene B4, but increased IL-1β production. Using specific compounds to inhibit the synthesis of each PG in vitro and in vivo, we found that endogenous PGD2 contributed to fungicidal mechanisms and controlled inflammation, whereas endogenous PGE2 decreased phagocytosis and killing of the fungus and induced inflammation. These findings demonstrate that, although PGD2 acts as an immunostimulatory mediator to control H. capsulatum infection, PGE2 has immunosuppressive effects, and the balance between these two PGs may limit collateral immune damage at the expense of microbial containment. Histoplasmosis is a fungal disease caused by Histoplasma capsulatum, mainly affecting the respiratory tract (1.Kauffman C.A. Histoplasmosis.Clin. Chest Med. 2009; 30: 217-225Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The incidence of histoplasmosis has increased worldwide, which is mostly associated with immunodeficiency, such as HIV (2.Kasuga T. White T.J. Koenig G. McEwen J. Restrepo A. Castaneda E. Lacaz C. Heins-Vaccari E.M. De Freitas R.S. Zancope-Oliveira R.M. et al.Phylogeography of the fungal pathogen Histoplasma capsulatum.Mol. Ecol. 2003; 12: 3383-3401Crossref PubMed Scopus (248) Google Scholar, 3.Colombo A.L. Tobon A. Restrepo A. Queiroz-Telles F. Nucci M. Epidemiology of endemic systemic fungal infections in Latin America.Med. Mycol. 2011; 49: 785-798PubMed Google Scholar, 4.Antinori S. Histoplasma capsulatum: more widespread than previously thought.Am. J. Trop. Med. Hyg. 2014; 90: 982-983Crossref PubMed Scopus (74) Google Scholar). In the lung, the immune response begins after the uptake of the fungus by alveolar macrophages (AMs) and dendritic cells. AMs have a special importance in lung defense, where they are responsible for protecting the alveolar epithelium by clearing microorganisms by phagocytosis and intracellular killing (5.Lohmann-Matthes M.L. Steinmuller C. Franke-Ullmann G. Pulmonary macrophages.Eur. Respir. 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Medeiros A.I. 5-Lipoxygenase deficiency impairs innate and adaptive immune responses during fungal infection.PLoS One. 2012; 7: e31701Crossref PubMed Scopus (38) Google Scholar), and regulate phagocytosis and antimicrobial activities of phagocytic cells (5.Lohmann-Matthes M.L. Steinmuller C. Franke-Ullmann G. Pulmonary macrophages.Eur. Respir. J. 1994; 7: 1678-1689Crossref PubMed Scopus (256) Google Scholar, 7.Secatto A. Rodrigues L.C. Serezani C.H. Ramos S.G. Dias-Baruffi M. Faccioli L.H. Medeiros A.I. 5-Lipoxygenase deficiency impairs innate and adaptive immune responses during fungal infection.PLoS One. 2012; 7: e31701Crossref PubMed Scopus (38) Google Scholar, 8.Pereira P.A. Trindade B.C. Secatto A. Nicolete R. Peres-Buzalaf C. Ramos S.G. Sadikot R. Bitencourt Cda S. Faccioli L.H. Celecoxib improves host defense through prostaglandin inhibition during Histoplasma capsulatum infection.Mediators Inflamm. 2013; 2013: 950981Crossref PubMed Scopus (24) Google Scholar). In particular, the synthesis and signaling of lipid mediators known as leukotrienes (LTs) and prostaglandins (PGs) are increased during histoplasmosis and further regulate host defense (8.Pereira P.A. Trindade B.C. Secatto A. Nicolete R. Peres-Buzalaf C. Ramos S.G. Sadikot R. Bitencourt Cda S. Faccioli L.H. Celecoxib improves host defense through prostaglandin inhibition during Histoplasma capsulatum infection.Mediators Inflamm. 2013; 2013: 950981Crossref PubMed Scopus (24) Google Scholar, 9.Medeiros A.I. Sa-Nunes A. Soares E.G. Peres C.M. Silva C.L. Faccioli L.H. Blockade of endogenous leukotrienes exacerbates pulmonary histoplasmosis.Infect. Immun. 2004; 72: 1637-1644Crossref PubMed Scopus (83) Google Scholar). Biosynthesis of PGs is coordinated by two distinct cyclooxygenase (COX) isoforms, the constitutive COX-1 and the inducible COX-2, which convert arachidonic acid (AA) to the unstable intermediate compound PGH2. Then, terminal synthase enzymes, including PGD and PGE synthases, generate PGD2 and PGE2, respectively, at sites of inflammation, resulting in either proinflammatory or anti-inflammatory effects, depending on the nature of the stimulus (10.Harris S.G. Padilla J. Koumas L. Ray D. Phipps R.P. Prostaglandins as modulators of immunity.Trends Immunol. 2002; 23: 144-150Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar, 11.Joo M. Sadikot R.T. PGD synthase and PGD2 in immune response.Mediators Inflamm. 2012; 2012: 503128Crossref PubMed Scopus (66) Google Scholar). Recently, we demonstrated that, during lethal H. capsulatum infection, pharmacological inhibition of COX-2 by the compound celecoxib increased mouse survival and the phagocytic capacity of AMs, suggesting a contribution of PGs to pathogenesis of this infection (8.Pereira P.A. Trindade B.C. Secatto A. Nicolete R. Peres-Buzalaf C. Ramos S.G. Sadikot R. Bitencourt Cda S. Faccioli L.H. Celecoxib improves host defense through prostaglandin inhibition during Histoplasma capsulatum infection.Mediators Inflamm. 2013; 2013: 950981Crossref PubMed Scopus (24) Google Scholar). Additionally, during bacterial and other fungal infections, PGE2 and PGD2 production is enhanced in the lung (8.Pereira P.A. Trindade B.C. Secatto A. Nicolete R. Peres-Buzalaf C. Ramos S.G. Sadikot R. Bitencourt Cda S. Faccioli L.H. Celecoxib improves host defense through prostaglandin inhibition during Histoplasma capsulatum infection.Mediators Inflamm. 2013; 2013: 950981Crossref PubMed Scopus (24) Google Scholar, 12.Joo M. Kwon M. Sadikot R.T. Kingsley P.J. Marnett L.J. Blackwell T.S. Peebles R.S. Urade Jr., Y. Christman J.W. Induction and function of lipocalin prostaglandin D synthase in host immunity.J. Immunol. 2007; 179: 2565-2575Crossref PubMed Scopus (39) Google Scholar). However, the specific roles of PGD2 and PGE2 during infections, especially in histoplasmosis, remain incompletely defined. Over time, PGE2 has emerged as a potent endogenous modulator of innate immunity and macrophage effector functions (10.Harris S.G. Padilla J. Koumas L. Ray D. Phipps R.P. Prostaglandins as modulators of immunity.Trends Immunol. 2002; 23: 144-150Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar, 13.Nakanishi M. Rosenberg D.W. Multifaceted roles of PGE2 in inflammation and cancer.Semin. Immunopathol. 2013; 35: 123-137Crossref PubMed Scopus (389) Google Scholar). Generally, PGE2 promotes endothelial cell-mediated vasodilatation and recruitment of circulating leukocytes during inflammation to areas of infection, through mechanisms activated by PGE2 binding to E prostanoid (EP) receptors (EP1–4) coupled to G proteins present in the cell membrane (13.Nakanishi M. Rosenberg D.W. Multifaceted roles of PGE2 in inflammation and cancer.Semin. Immunopathol. 2013; 35: 123-137Crossref PubMed Scopus (389) Google Scholar). On the other hand, PGE2 inhibits macrophage effector mechanisms, such as phagocytosis, through EP2 receptor and bacterial killing through EP2–4 receptors, both coupled to Gαs proteins, leading to activation of adenylate cyclase, which increases cyclic adenosine monophosphate (cAMP) concentrations (14.Serezani C.H. Chung J. Ballinger M.N. Moore B.B. Aronoff D.M. Peters-Golden M. Prostaglandin E2 suppresses bacterial killing in alveolar macrophages by inhibiting NADPH oxidase.Am. J. Respir. Cell Mol. Biol. 2007; 37: 562-570Crossref PubMed Scopus (128) Google Scholar, 15.Aronoff D.M. Canetti C. Peters-Golden M. Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP.J. Immunol. 2004; 173: 559-565Crossref PubMed Scopus (284) Google Scholar, 16.Zoccal K.F. Sorgi C.A. Hori J.I. Paula-Silva F.W.G. Arantes E.C. Serezani C.H. Zamboni D.S. Faccioli L.H. Opposing roles of LTB4 and PGE2 in regulating the inflammasome-dependent scorpion venom-induced mortality.Nat. Commun. 2016; 7: 10760Crossref PubMed Scopus (71) Google Scholar) and IL-1β production (16.Zoccal K.F. Sorgi C.A. Hori J.I. Paula-Silva F.W.G. Arantes E.C. Serezani C.H. Zamboni D.S. Faccioli L.H. Opposing roles of LTB4 and PGE2 in regulating the inflammasome-dependent scorpion venom-induced mortality.Nat. Commun. 2016; 7: 10760Crossref PubMed Scopus (71) Google Scholar). PGD2, in turn, binds to the D prostanoid receptor 1 (DP1) and to the "chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes" receptor (DP2 or CRTH2) (17.Hirai H. Tanaka K. Yoshie O. Ogawa K. Kenmotsu K. Takamori Y. Ichimasa M. Sugamura K. Nakamura M. Takano S. et al.Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2.J. Exp. Med. 2001; 193: 255-261Crossref PubMed Scopus (964) Google Scholar). PGD2 binds to DP1, a transmembrane receptor coupled to the G-protein subunit Gαs. This prostanoid also induces elevation of cAMP, resulting in the inhibition of effector mechanisms of macrophages and other cells (18.Jandl K. Stacher E. Balint Z. Sturm E.M. Maric J. Peinhaupt M. Luschnig P. Aringer I. Fauland A. Konya V. et al.Activated prostaglandin D2 receptors on macrophages enhance neutrophil recruitment into the lung.J. Allergy Clin. Immunol. 2016; 137: 833-843Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 19.Chen Y. Perussia B. Campbell K.S. Prostaglandin D2 suppresses human NK cell function via signaling through D prostanoid receptor.J. Immunol. 2007; 179: 2766-2773Crossref PubMed Scopus (41) Google Scholar, 20.Faveeuw C. Gosset P. Bureau F. Angeli V. Hirai H. Maruyama T. Narumiya S. Capron M. Trottein F. Prostaglandin D2 inhibits the production of interleukin-12 in murine dendritic cells through multiple signaling pathways.Eur. J. Immunol. 2003; 33: 889-898Crossref PubMed Scopus (58) Google Scholar). However, when PGD2 binds to DP2, a Gαi protein subunit coupled receptor, it reduces cAMP concentrations (21.Sawyer N. Cauchon E. Chateauneuf A. Cruz R.P. Nicholson D.W. Metters K.M. O'Neill G.P. Gervais F.G. Molecular pharmacology of the human prostaglandin D2 receptor, CRTH2.Br. J. Pharmacol. 2002; 137: 1163-1172Crossref PubMed Scopus (171) Google Scholar). Consequently, the potentially contrasting immunoregulatory roles of PGD2 and PGE2 require further investigation, which is the focus of the present research. Mammalian cells communicate with each other by exchanging signals that bind specifically to surface or intracellular receptors, followed by a cascade of events that amplify and transduce the incoming signal and eventually elicit a cellular response. The cAMP-dependent protein kinase A (PKA), mitogen-activated protein kinase (MAPK), and nuclear factor κB (NF-κB) cascades modulate common processes in the cell, and multiple levels of cross-talk between these signaling pathways have been described (22.Ubersax J.A. Ferrell Jr, J.E. Mechanisms of specificity in protein phosphorylation.Nat. Rev. Mol. Cell Biol. 2007; 8: 530-541Crossref PubMed Scopus (995) Google Scholar, 23.Han J. Ulevitch R.J. Limiting inflammatory responses during activation of innate immunity.Nat. Immunol. 2005; 6: 1198-1205Crossref PubMed Scopus (263) Google Scholar). Because activation of PG receptors/cAMP axes is important to regulate macrophage effector functions (14.Serezani C.H. Chung J. Ballinger M.N. Moore B.B. Aronoff D.M. Peters-Golden M. Prostaglandin E2 suppresses bacterial killing in alveolar macrophages by inhibiting NADPH oxidase.Am. J. Respir. Cell Mol. Biol. 2007; 37: 562-570Crossref PubMed Scopus (128) Google Scholar, 15.Aronoff D.M. Canetti C. Peters-Golden M. Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP.J. Immunol. 2004; 173: 559-565Crossref PubMed Scopus (284) Google Scholar), the putative role of PGD2 and PGE2 in cell signaling activation and inflammatory mediator synthesis deserves detailed examination. Despite the fact that PGE2 has been shown to inhibit phagocytosis and killing of pathogens by macrophages (14.Serezani C.H. Chung J. Ballinger M.N. Moore B.B. Aronoff D.M. Peters-Golden M. Prostaglandin E2 suppresses bacterial killing in alveolar macrophages by inhibiting NADPH oxidase.Am. J. Respir. Cell Mol. Biol. 2007; 37: 562-570Crossref PubMed Scopus (128) Google Scholar, 15.Aronoff D.M. Canetti C. Peters-Golden M. Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP.J. Immunol. 2004; 173: 559-565Crossref PubMed Scopus (284) Google Scholar), the role of PGD2 in modulating AM effector functions is not known. In the present study, we determined the contribution of endogenous and exogenous PGD2 and PGE2 on AM effector functions after immune serum (IS)-opsonized H. capsulatum (Ops-H. capsulatum) infection. Using rat AMs, we found that endogenous and exogenous PGD2 and PGE2 actions result in divergent effects on phagocytosis and fungicidal activities of these cells. Treatment of Ops-H. capsulatum-infected AMs with exogenous PGD2 increased phagocytosis and killing through the DP2 receptor; it also inhibited phosphorylation of phospholipase C-γ (PLC-γ) without affecting c-Jun N-terminal kinase 1/2 (JNK1/2), p38, and NF-κB. Furthermore, PGD2 inhibited IL-10 production by infected AMs, while increasing the expression of high-affinity receptors for LTB4 (BLT1). An opposite effect was observed for PGE2, which reduced phagocytosis and killing through an EP2/EP4-dependent manner. Additionally, it diminished phosphorylation of PLC-γ, p38, and NF-κB, while amplifying JNK1/2, and reducing TNF-α, H2O2, and LTB4 production by Ops-H. capsulatum-infected AMs. Finally, in vivo experiments demonstrated that inhibition of PGD2 synthesis increased susceptibility to infection in mice, as well as increased inflammatory cytokine production, while inhibition of PGE2 synthesis increased resistance against infection and diminished lung tissue damage. Also, endogenous PGD2 regulated only fungicidal mechanisms, whereas endogenous PGE2 coordinated phagocytosis and killing of the fungus by AMs. Given the opposite effects of PGD2 and PGE2 during AM infection, these lipids might be potential targets to treat fungal lung diseases, particularly histoplasmosis. Pathogen-free male Wistar rats (125–150 g) and male C57BL/6 mice (20–22 g) were obtained from the animal facilities of the Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo. All experiments were approved and conducted in accordance with the guidelines of the Animal Care Committee of the University of São Paulo (Protocols 09.1.375.53.5 and 013.2016-1). The in vitro experiments with H. capsulatum-infected AMs and infected animals were performed in level 3 biohazard facilities at Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo. The H. capsulatum clinical isolate was obtained from a patient at the Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo. The mycelia were obtained by culturing fungi at 25°C in Sabouraud dextrose agar tubes (Difco, Detroit, MI), and the live yeast fungus was subcultured at 37°C on glutamine-cysteine-sheep blood (5%) BHI (Detroit, MI) for 15 days. Yeast cells were used when their viability was ≥90% according to fluorescein diacetate (Sigma-Aldrich, St. Louis, MO) and ethidium bromide (Sigma-Aldrich) staining (8.Pereira P.A. Trindade B.C. Secatto A. Nicolete R. Peres-Buzalaf C. Ramos S.G. Sadikot R. Bitencourt Cda S. Faccioli L.H. Celecoxib improves host defense through prostaglandin inhibition during Histoplasma capsulatum infection.Mediators Inflamm. 2013; 2013: 950981Crossref PubMed Scopus (24) Google Scholar, 9.Medeiros A.I. Sa-Nunes A. Soares E.G. Peres C.M. Silva C.L. Faccioli L.H. Blockade of endogenous leukotrienes exacerbates pulmonary histoplasmosis.Infect. Immun. 2004; 72: 1637-1644Crossref PubMed Scopus (83) Google Scholar). Rats were intraperitoneally inoculated with 1 ml containing 108 yeast of H. capsulatum, and 10 days later were submitted to a second inoculation with an equal inoculum. After 7 days, the rats were decapitated, and blood was collected and centrifuged at 1,900 g for 10 min to obtain the IS. IS was heated at 56°C for 1 h to inactivate complement proteins and stored at −80°C (24.Mancuso P. Peters-Golden M. Modulation of alveolar macrophage phagocytosis by leukotrienes is Fc receptor-mediated and protein kinase C-dependent.Am. J. Respir. Cell Mol. Biol. 2000; 23: 727-733Crossref PubMed Scopus (40) Google Scholar). For fungus opsonization, 1 × 108 yeast in 1 ml of PBS was incubated with 10% IS for 30 min at 37°C on a rotating platform (7.Secatto A. Rodrigues L.C. Serezani C.H. Ramos S.G. Dias-Baruffi M. Faccioli L.H. Medeiros A.I. 5-Lipoxygenase deficiency impairs innate and adaptive immune responses during fungal infection.PLoS One. 2012; 7: e31701Crossref PubMed Scopus (38) Google Scholar), and the opsonized fungus is referred to as Ops-H. capsulatum. Nonopsonized fungi (H. capsulatum) incubated only with PBS were used for comparison as described. Resident AMs from naïve rats were obtained via ex vivo lung lavage (24.Mancuso P. Peters-Golden M. Modulation of alveolar macrophage phagocytosis by leukotrienes is Fc receptor-mediated and protein kinase C-dependent.Am. J. Respir. Cell Mol. Biol. 2000; 23: 727-733Crossref PubMed Scopus (40) Google Scholar) and suspended in incomplete RPMI 1640 at 2 × 106 cells per ml. Cells were allowed to adhere to tissue culture plates for 1 h (37°C, 5% CO2), followed by two washes with warm incomplete RPMI 1640, resulting in ± 99% of adherent cells identified as AMs by staining with Panoptic (Laborclin, Paraná, Brazil). Cells were cultured overnight in complete RPMI 1640 containing 10% FBS and 1% penicillin/streptomycin/amphotericin B (Gibco, Grand Island, NY). The following day, cells were washed twice with warm medium (incomplete RPMI 1640) to remove nonadherent cells. AMs were preincubated with indomethacin (10 μM) (COX1/2 inhibitor; Sigma-Aldrich), or celecoxib (10 μM) (COX-2 inhibitor; Celebra®, Pfizer, SP, Brazil), or HQL-79 (1 μM) (PGD2 synthase inhibitor); or CAY10526 (1 μM) (PGE2 synthase inhibitor); or BWA868c (1 μM) (DP1 antagonist); or Bay-u3405 (1 μM) (DP2 antagonist); or AH6809 (1 μM) (EP2 antagonist); or AH23848 (1 μM) (EP4 antagonist), preceding fungus infection. The enzyme inhibitors or antagonists were purchased from Cayman Chemical (Ann Arbor, MI), and incubated with AMs for 30 min before infection. When necessary, cells were incubated with PGD2 or PGE2 for 2 min (1 μM) (Cayman Chemical), before in vitro infection. Receptor antagonist and concentrations used were previously determined by our group (16.Zoccal K.F. Sorgi C.A. Hori J.I. Paula-Silva F.W.G. Arantes E.C. Serezani C.H. Zamboni D.S. Faccioli L.H. Opposing roles of LTB4 and PGE2 in regulating the inflammasome-dependent scorpion venom-induced mortality.Nat. Commun. 2016; 7: 10760Crossref PubMed Scopus (71) Google Scholar, 25.Assis P.A. Espindola M.S. Paula-Silva F.W. Rios W.M. Pereira P.A. Leao S.C. Silva C.L. Faccioli L.H. Mycobacterium tuberculosis expressing phospholipase C subverts PGE2 synthesis and induces necrosis in alveolar macrophages.BMC Microbiol. 2014; 14: 128Crossref PubMed Scopus (26) Google Scholar) or investigated in the literature (14.Serezani C.H. Chung J. Ballinger M.N. Moore B.B. Aronoff D.M. Peters-Golden M. Prostaglandin E2 suppresses bacterial killing in alveolar macrophages by inhibiting NADPH oxidase.Am. J. Respir. Cell Mol. Biol. 2007; 37: 562-570Crossref PubMed Scopus (128) Google Scholar, 15.Aronoff D.M. Canetti C. Peters-Golden M. Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP.J. Immunol. 2004; 173: 559-565Crossref PubMed Scopus (284) Google Scholar, 26.Wang J.W. Woodward D.F. Martos J.L. Cornell C.L. Carling R.W. Kingsley P.J. Marnett L.J. Multitargeting of selected prostanoid receptors provides agents with enhanced anti-inflammatory activity in macrophages.FASEB J. 2016; 30: 394-404Crossref PubMed Scopus (12) Google Scholar, 27.Aritake K. Kado Y. Inoue T. Miyano M. Urade Y. Structural and functional characterization of HQL-79, an orally selective inhibitor of human hematopoietic prostaglandin D synthase.J. Biol. Chem. 2006; 281: 15277-15286Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Incomplete RPMI 1640 containing the same concentrations of DMSO and/or alcohol used to dissolve the compounds was used as control and identified as vehicle. Cells treated with compounds or vehicle were used for phagocytic and fungicidal assays. A fluorometric phagocytosis assay was performed to test the capacity of AMs to phagocytize FITC-labeled H. capsulatum (or Ops-H. capsulatum) as published previously (7.Secatto A. Rodrigues L.C. Serezani C.H. Ramos S.G. Dias-Baruffi M. Faccioli L.H. Medeiros A.I. 5-Lipoxygenase deficiency impairs innate and adaptive immune responses during fungal infection.PLoS One. 2012; 7: e31701Crossref PubMed Scopus (38) Google Scholar). Briefly, yeast cells were labeled with FITC (Amresco, OH) for 1 h at 37°C (7.Secatto A. Rodrigues L.C. Serezani C.H. Ramos S.G. Dias-Baruffi M. Faccioli L.H. Medeiros A.I. 5-Lipoxygenase deficiency impairs innate and adaptive immune responses during fungal infection.PLoS One. 2012; 7: e31701Crossref PubMed Scopus (38) Google Scholar). FITC-labeled Ops-H. capsulatum or FITC-labeled H. capsulatum was added, and the number of yeast cells to be used by AMs was determined through multiplicity of infection (MOI) starting from 1:1; 1:5, or 1:10, respectively. The AMs were pretreated or not with the compounds as described above before the fungus was added and then incubated in the dark (37°C, 5% CO2). After 2 h, free yeast cells were removed by washing with warm sterile PBS, and the residual extracellular FITC was quenched with Trypan blue (250 mg/ml; Gibco) for 1 min. Fluorescence was determined by using a micro plate reader (485 nm excitation/535 nm emission, SPECTRAMax, Molecular Devices, Sunnyvale, CA). Phagocytosis was determined by the mean of relative fluorescence units (MFI) emitted from intracellular fungi. AMs were pretreated with IFN-γ (50 ng/ml) overnight to improve their effector mechanism as described by Peck (28.Peck R. A one-plate assay for macrophage bactericidal activity.J. Immunol. Methods. 1985; 82: 131-140Crossref PubMed Scopus (84) Google Scholar), and submitted or not to the above treatments. Next, cells were incubated with H. capsulatum (opsonized or not) at MOI 1:10, and after 2 h, cells were washed twice with warm sterile PBS to remove the extracellular yeasts. Following another 48 h of incubation, the supernatants were collected and kept at −80°C until they were used for measurements, as described. Afterward, the cells were lysed by adding 200 µl of 0.05% saponin, and an aliquot was plated on BHI agar-blood (29.Brummer E. Stevens D.A. Antifungal mechanisms of activated murine bronchoalveolar or peritoneal macrophages for Histoplasma capsulatum.Clin. Exp. Immunol. 1995; 102: 65-70Crossref PubMed Scopus (37) Google Scholar). After 21 days of culture at 37°C, the colony-forming units (CFU) were counted and the fungicidal activity was calculated according to the formula: [100 - (100 × CFU experimental)/CFU control]. AMs treated or not with the above compounds were incubated with Ops-H. capsulatum, and after 48 h the cell culture supernatants or lung homogenate of H. capsulatum-infected mice were obtained to measure TNF-α, IL-1β, and IL-10, by using commercially available ELISA kits (R&D Systems, Minneapolis, MN). For each sample, the cytokine concentrations were obtained from a standard curve established with the appropriate recombinant cytokine. The sensitivities were >10 pg/ml. The release of H2O2 from AMs was determined by phenol red oxidation, as described previously (30.Pick E. Charon J. Mizel D. A rapid densitometric microassay for nitroblue tetrazolium reduction and application of the microassay to macrophages.J. Reticuloendothel. Soc. 1981; 30: 581-593PubMed Google Scholar), in supernatant from cells pretreated or not with PGD2 or PGE2, and incubated with Ops-H. capsulatum (MOI 1:10). After 48 h of incubation, the supernatants were replaced by supplemented assay medium (RPMI 1640-containing peroxidase and phenol red) and incubated for 2 h. Then, stop solution (NaOH 1N) was added, and optical density (OD) was determined at 620 nm. The expression of BLT1 was determined by using the flow-cytometry immune staining protocol with antibodies conjugated with fluorochromes (BD Biosciences, Franklin Lakes, NJ). The AMs (2 × 105 cells) were pretreated with the compounds described above before the fungus was added (2 × 104 cells) and incubated at 37°C, 5% CO2. After 2 h, the supernatant was removed, and the cells were washed with 300 µl of PBS containing 2% FBS (Gibco). Cells were then suspended in polystyrene tubes and centrifuged at 400 g for 5 min. Cells were suspended in Fc block (200 µl), containing anti-CD16/CD32 antibodies at dilution 1:100 and incubated for 30 min at 4°C. Subsequently, the antibody against BLT1 was added to the AMs. After 40 min, cells were washed twice, and then fixed with PBS containing 1% (wt/vol) paraformaldehyde. A total of 20,000 events was acquired for each tube (FACSCantoTM; Becton Dickinson), using the FACSDiva software. Eicosanoids were purified from the supernatant of AMs cultures or lung homogenate (Mixer Homogenizer, IKA, Wilmington, NC) by using Sep-Pak C18 cartridges according to the manufacturer's instructions (Waters Corp.. Milford, MA). Quantifications of PGE2 and LTB4 (Enzo Life Science, Farmingdale, NY) and PGD2 (Cayman Chemical) were performed by using specific enzyme immunoassay kits according to manufacturers' instructions, and the results were expressed in picograms per milliliter, or, after the data were transformed, as percentages, and infected AM was set as 100%. Samples of AMs were prepared as previously described (31.Zoccal K.F. Bitencourt Cda S. Paula-Silva F.W. Sorgi C.A. de Castro Figueiredo Bordon K. Arantes E.C. Faccioli L.H. TLR2, TLR4 and CD14 recognize venom-associated molecular patterns from Tityus serrulatus to induce macrophage-derived inflammatory mediators.PLoS One. 2014; 9: e88174Crossref PubMed Scopus (59) Google Scholar). After infection and treatments, cells were washed with ice-cold PBS and then lysed with buffer containing protease and phosphatase inhibitors according to the manufacturer's protocol for adherent cells (Becton Dic
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