Microbes and Infection 9 (2007) 744e747 www.elsevier.com/locate/micinf
Short communication
Prostaglandin E2 inhibits Paracoccidioides brasiliensis killing by human monocytes Ana Paula Bordon a, Luciane Alarc~ao Dias-Melicio a, Michele Janegitz Acorci a, Sueli Aparecida Calvi b, Maria Terezinha Serr~ao Perac¸oli a, Angela Maria Victoriano de Campos Soares a,* a
Department of Microbiology and Immunology, Institute of Biosciences, S~ao Paulo State University e UNESP, Botucatu, CEP: 18618-000 Sao Paulo, Brazil b Department of Tropical Diseases and Image Diagnosis, Botucatu Medical School, S~ao Paulo State University e UNESP, Botucatu, CEP: 18618-000 Sao Paulo, Brazil Received 22 June 2006; accepted 17 February 2007 Available online 24 February 2007
Abstract Human monocytes lacked fungicidal activity against high virulence strain of Paracoccidioides brasiliensis, even after IFN-g activation. However, monocytes treated with indomethacin exhibited an effective killing against this fungus, suggesting a role of prostaglandin E2 (PGE2) in the inhibition process. Thus, the purpose of this work was to determine whether the effect of PGE2 in fungicidal activity was related with decrease on H2O2 release, the metabolite involved in P. brasiliensis killing, and changes in the levels of TNF-a, IL-6 and IL-10. Human monocytes challenged with the fungus produced high PGE2 levels, which in turn inhibited the fungicidal activity of these cells by reducing H2O2 and TNFa production. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Prostaglandin E2; H2O2; Cytokines; Monocytes; Paracoccidioides brasiliensis
1. Introduction Paracoccidioidomycosis is a deep mycosis which is endemic in Latin America. This disease is caused by Paracoccidioides brasiliensis, a fungus that undergoes thermal dimorphism, developing as yeast at body temperature [1]. Since the etiological agent is considered an intracellular pathogen, studies of its interaction with phagocytic cells are essential for the understanding of the hosteparasite relationship. However, the modulation of the mononuclear phagocyteeP. brasiliensis interactions has not been fully elucidated. Previous study showed the lack of fungicidal activity by human monocytes, even after IFN-g activation. These results were associated to the fungus capacity to * Corresponding author. Tel.: þ55 015 14 3811 6058; fax: þ55 015 14 3815 3744. E-mail address:
[email protected] (A.M. Victoriano de Campos Soares). 1286-4579/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2007.02.019
induce the release of prostaglandins, given that a significant fungicidal activity was detected after monocytes treatment with indomethacin (INDO), a cyclooxygenase inhibitor [2]. Thus, the purpose of this work was to determine the capacity of P. brasiliensis to induce PGE2 release by human monocytes and if this mediator suppresses fungicidal activity by decreasing H2O2 release, the metabolite involved in P. brasiliensis killing by human monocytes. Moreover, the effect of this mediator on the levels of cytokines, such as TNF-a, IL-6 and IL-10, was also evaluated. 2. Materials and methods 2.1. Donors This study included 20 healthy blood donors from the University Hospital of the Botucatu Medical School, S~ao Paulo State University, Brazil (aged 20e50 years). Approval from
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the Ethics Committee of the Botucatu Medical School was obtained.
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Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by density gradient centrifugation with HistopaqueÒ e1077 (Sigma-Aldrich, Inc., St. Louis, MO, USA). The PBMC suspension was stained with neutral red (0.02%) that is incorporated by monocytes and allows their identification and counting. Then, the mononuclear cells were suspended to 2 106 monocytes/ml in RPMI-1640 (SigmaAldrich) complete tissue culture medium (CTCM) [2] and dispensed into 100 ml/well in 96-well flat-bottom plates (Nunc, Life Tech. Inc., MD, USA) for fungicidal activity and H2O2 production. The suspensions containing 1 106 monocytes/ ml were dispensed into 1000 ml/well in 24-well flat-bottomed plates (Nunc) for culture supernatant cytokines measurement assays. After incubation for 2 h at 37 C in 5% CO2, nonadherent cells from both plates were removed and each well was rinsed twice with RPMI-1640. After adherence, the monocytes were treated at 37 C in 5% CO2 for 18 h with the following stimuli: (i) IFN-g 500 U/ml (R&D Systems, Minneapolis, MN, USA), (ii) INDO 20 mg/ml (Sigma-Aldrich), (iii) IFN-g 500 U/ml plus INDO 20 mg/ml. 2.4. Fungicidal activity and quantification of H2O2, PGE2 and cytokines After treatment, culture supernatants were removed from both plates and monolayers were challenged with a suspension containing 4 104 yeasts/ml (ratio of 1 fungus:50 monocytes) prepared in CTCM plus 10% fresh human AB serum in 5% CO2 at 37 C. After 4 h, the cells were evaluated for fungicidal activity [2] and H2O2 release [3], and supernatants for PGE2, TNF-a, IL-6 and IL-10 concentrations by ELISA (R&D Systems Kits). 2.5. Statistical analysis The results were compared by analysis of variance (ANOVA) followed by the Tukey test ( p < 0.05). 3. Results 3.1. Effect of PGE2 on the fungicidal activity of human monocytes Fig. 1A shows that nonactivated cells and even IFN-gactivated monocytes lacked fungicidal activity against Pb18. However, when human monocytes were treated with
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The high virulence P. brasiliensis strain 18 (Pb18) was used throughout this study. Yeast viability was determined [2] and suspensions containing more than 90% viable yeasts were used.
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Fig. 1. Fungicidal activity (A), PGE2 (B) and H2O2 production (C) by human monocytes pretreated with IFN-g (500 U/ml), INDO (20 mg/ml) or IFN-g (500 U/ml) plus INDO (20 mg/ml) for 18 h, and either challenged - or not , with P. brasiliensis strain 18 (Pb18) for 4 h. Results are expressed as mean SEM and were obtained from monocyte cocultures of 20 healthy donors. *p < 0.05 CTCM; þp < 0.05 CTCM þ Pb18.
INDO, significant fungicidal activity was detected. This activity was slightly increased after cells preincubation with INDO plus IFN-g, but the differences were not significant statistically. The INDO concentration was previously standardized in our laboratory for inducing higher fungicidal
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activity, and in our control studies this drug did not affect the viability of the cells.
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In order to test whether the inhibitory effect of PGE2 correlated with a decrease in H2O2 levels, the production of this metabolite was evaluated in all tested cultures. Fig. 1C shows that both nonactivated and INDO-treated monocytes released low H2O2 levels. However, when IFN-g, or INDO plus IFN-g were used, H2O2 levels were significantly increased. Nonactivated monocytes challenged with Pb18 released low levels of H2O2, whose production slightly increased when monocytes were IFN-g preactivated. However, a significant increase in H2O2 levels was obtained only when monocytes were incubated with INDO and INDO plus IFN-g. These results show that PGE2 release in fungus-challenged cultures was associated with low H2O2 levels.
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In order to assess whether the PGE2 inhibitory effect on fungicidal activity and H2O2 release was associated with changes in the production of important immunoregulatory cytokines, TNF-a, IL-6 and IL-10 levels were determined. In culture supernatants of nontreated monocytes, low levels of TNF-a were detected whereas when these cells were treated with IFN-g, or INDO plus IFN-g, TNF-a levels were significantly increased. These levels remained unchanged when INDO treatment was employed (Fig. 2A). After challenge, higher TNF-a levels were released after INDO and INDO plus IFN-g treatments when compared to their respective nonchallenged cultures. These results indicate that PGE2 levels inhibited TNF-a production by nonactivated and even IFN-g activated monocytes. IL-6 and IL-10 production are shown in Fig. 2B and C, respectively. The monocytes not challenged with Pb18 and cultivated with different stimuli, produced low levels of IL-6 and IL-10. In the monocyte cultures challenged with Pb18, nonactivated monocytes released higher IL-6 levels when compared
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3.4. Effect of prostaglandins on TNF-a, IL-6 and IL-10 production by human monocytes
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3.2. PGE2 production by human monocytes In order to attest that the inhibitory effect on fungicidal activity was PGE2 related, the levels of this mediator were evaluated. Nontreated monocytes produced low PGE2 levels, which were increased when monocytes were preactivated with IFN-g. PGE2 levels were significantly inhibited when cells were preincubated with INDO plus IFN-g (Fig. 1B). As expected, PGE2 release was greater in all fungus-challenged cultures than in nonchallenged cultures. However, PGE2 levels were significantly inhibited when monocytes were incubated with either INDO or INDO plus IFN-g.
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Fig. 2. TNF-a (A), IL-6 (B) and IL-10 (C) production by human monocytes pretreated with IFN-g (500 U/ml), INDO (20 mg/ml) or IFN-g (500 U/ml) plus INDO (20 mg/ml) for 18 h, and either challenged - or not , with P. brasiliensis strain 18 (Pb18) for 4 h. Results are expressed as mean SEM and were obtained from monocyte cocultures of 20 healthy donors. *p < 0.05 CTCM; þp < 0.05 CTCM þ Pb18.
to nonchallenged ones, indicating the capacity of Pb18 to stimulate human cells to produce this cytokine. However, no change in IL-6 production was observed when treatment with IFN-g, INDO or INDO plus IFN-g was used, suggesting that PGE2 does not affect the levels of this cytokine.
A.P. Bordon et al. / Microbes and Infection 9 (2007) 744e747
Similar results were obtained when IL-10 was assayed (Fig. 2C). 4. Discussion Our results showed that the inhibitory effect of PGE2 on monocytes fungicidal activity, even after IFN-g activation, was associated with low levels of H2O2, the metabolite involved in P. brasiliensis killing by activated human monocytes [3]. This strongly suggests that P. brasiliensis escapes from phagocytic cells activity by inducing PGE2. These findings are in agreement with other studies showing that, to evade host defenses, important pathogenic fungi develop several mechanisms, such as oxidative metabolism inhibition [4e7]. Moreover, the inhibitory effect of PGE2 on phagocytic cells functions has been observed in the interaction between these cells and other microorganisms, such as Mycobaterium sp. [8,9]. This study also investigated whether PGE2 inhibition was associated with changes in the levels of TNF-a, IL-6 and IL-10 in cocultures supernatants. The levels of TNF-a were higher in the supernatants of INDO and INDO plus IFN-g-treated cells, suggesting that PGE2 acts on H2O2 release by inhibiting TNF-a production. This effect may result in deficient monocyte activation with consequent release of low levels of this metabolite. This process may better explain P. brasiliensis killing by INDO and INDO plus IFN-g treated cells. Our data on TNF-a production are in agreement with the literature, given that several papers have shown PGE2 capacity to inhibit the production as well as the receptor expression of this cytokine [10]. In this study, P. brasiliensis was also found to stimulate human monocytes to release high levels of both IL-6 and IL-10, which, contrarily to TNF-a, were not affected by treatment with either INDO or INDO plus IFN-g, suggesting that PGE2 is not involved in the production of these cytokines after the challenge of human monocytes with P. brasiliensis. These results were not expected, since some studies have demonstrated that the inhibitory effect of PGE2 upon TNFa production would be mediated by IL-6, and that the same would apply for IL-10, as several works have shown the stimulating effect of PGE2 on this cytokine production [11e13]. However, in this study, the results obtained did not allow us to consider the inhibitory effect of PGE2 on TNF-a production to be associated with its capacity to upregulate IL-6 and IL-10. Although PGE2 did not modulate the levels of these cytokines, we must consider that IL-10 may have a role on killing inhibition. Previous studies on our laboratory have been shown that IL-10, but not TGF-b blocks the effect of TNF-a-activated monocytes and neutrophils [14,15]. Our findings together showed that the suppressor effect of PGE2 on a high virulence strain of P. brasiliensis killing by human monocytes is associated with its capacity to downregulate TNF-a and H2O2 synthesis.
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Acknowledgements This study was supported by grants from Fundac¸~ao de Amparo a` Pesquisa do Estado de S~ao Paulo (FAPESP), process no 02/06618-1, Brazil. References [1] A. Restrepo, A.M. Tobo´n, in: G.L. Mandell, J.E. Bennett, R. Dollin (Eds.), Paracoccidioides brasiliensis. Principles and Practice of Infectious Diseases, Elsevier, Philadelphia, 2005, pp. 3062e3068. [2] A.M.V.C. Soares, S.A. Calvi, M.T.S. Perac¸oli, A.C. Fernandez, L.A. Dias, A.R. Anjos, Modulatory effect of prostaglandins on human monocyte activation for killing of high- and low-virulence strains of Paracoccidioides brasiliensis, Immunol. 102 (2001) 480e485. [3] J.P.M. Carmo, L.A. Dias-Melicio, S.A. Calvi, M.T.S. Perac¸oli, A.M.V.C. Soares, TNF-a activates human monocytes for Paracoccidioides brasiliensis killing by an H2O2-dependent mechanism, Med. Mycol. 44 (2006) 363e368. [4] T. Ikeda, J.R. Little, Deactivation of macrophage oxidative burst in vitro by different strains of Histoplasma capsulatum, Mycopathology 132 (1995) 133e141. [5] M.C. Noverr, G.M. Cox, J.R. Perfect, G.B. Huffnagle, Role of PLB1 in pulmonary inflammation and cryptococcal eicosanoid production, Infect. Immun. 71 (2003) 1538e1547. [6] S. Tsunawaki, L.S. Yoshida, S. Nishida, T. Kobayashi, T. Shimoyama, Fungal metabolite gliotoxin inhibits assembly of the human respiratory burst NADPH oxidase, Infect. Immun. 72 (2004) 3373e3382. [7] C. Fradin, P. De Groot, D. Maccallum, M. Schaller, F. Klis, F.C. Odds, B. Hubbe, Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood, Mol. Microbiol. 56 (2005) 397e415. [8] N. Rastogi, M. Bachelet, J.P. Carvalho de Souza, Intracellular growth of Mycobacterium avium in human macrophages is linked to the increased synthesis of prostaglandin E2 and inhibition of the phagossomeelysossome fusions, FEMS Microbiol. Immunol. 4 (1992) 273e279. [9] N. Venkataprasad, H. Shiratsuchi, J.L. Jonson, Induction of prostaglandin E2 by human monocytes infected with Mycobacterium avium complex e modulation of cytokine expression, J. Infect. Dis. 174 (1996) 806e811. [10] S. Shinomiya, H. Naraba, A. Ueno, I. Utsunomiya, T. Maruyama, S. Ohuchida, F. Ushikubi, K. Yuki, S. Narumiya, Y. Sugimoto, A. Ichikawa, S. Oh-ishi, Regulation of TNF-alpha and interleukin-10 production by prostaglandins I(2) and E(2): studies with prostaglandin receptor-deficient mice and prostaglandin E-receptor subtype-selective synthetic agonists, Biochem. Pharmacol. 61 (2001) 1153e1160. [11] G. Strassmann, V. Patil-Koota, F. Finkelman, M. Fong, T. Kambayashi, Evidence for the involvement of interleukin-10 in the differential deactivation or murine peritoneal macrophage by prostaglandin E2, J. Exp. Med. 180 (1994) 2365e2370. [12] S. Berger, H. Ballo, H.J. Stutte, Immune complex-induced interleukin-6, interleukin-10 and prostaglandin secretion by human monocytes: a network of pro- and anti-inflammatory cytokines dependent the antigen:antibody ratio, Eur. J. Immunol. 26 (1996) 1297e1301. [13] Y. Niho, H. Niiro, H. Tanaka, H. Nakashima, T. Otsuk, Role of IL-10 in the crossregulation of prostaglandins and cytokines in monocytes, Acta Haematol. 99 (1998) 165e170. [14] A.M.V.C. Soares, W.B. Silva, D.R. Rodrigues, S.A. Calvi, C.S. Kurokawa, M.T.S. Perac¸oli, IL-10 but not TGF-b inhibits Paracoccidioides brasiliensis killing by human activated monocytes, Annu. Rev. Biomed. Sci. SI. (2002) 89. [15] D.L. Costa, L.A. Dias-Melicio, M.J. Acorci, A.P. Bordon, E.G. Tavian, M.T.S. Perac¸oli, A.M.V.C. Soares, Effect of interleukin-10 on the Paracoccidioides brasiliensis killing by gamma-interferon activated human neutrophils, Microbiol. Immunol. 51 (2007) 73e80.
