Respiratory Burst Responses of Rat Macrophages to Microsporidian Spores

Experimental Parasitology 98, 1–9 (2001) doi:10.1006/expr.2001.4610, available online at http://www.idealibrary.com on

Respiratory Burst Responses of Rat Macrophages to Microsporidian Spores

J. Leiro,*,1 R. Iglesias,† A. Parama´,† M. L. Sanmartin,† and F. M. Ubeira* *Departamento de Microbiologı´a y Parasitologı´a and †Instituto de Investigacio´n y Ana´lisis Alimentarios, Laboratorio de Parasitologı´a, Facultad de Farmacia, Universidad de Santiago de Compostela, 15706 Santiago de Compostela, Spain

Leiro, J., Iglesias, R., Parama´, A., Sanmartin, M. L., and Ubeira, F. M. 2001. Respiratory burst responses of rat macrophages to microsporidian spores. Experimental Parasitology 98, 1–9. This study investigated the respiratory burst responses of rat resident peritoneal macrophages and of peritoneal macrophages stimulated 5 days previously with viable spores of the fish infecting microsporidian Microgemma caulleryi. Nitric oxide production by resident macrophages and prestimulated macrophages in response to viable microsporidian spores was significantly lower than in response to Escherichia coli lipopolysaccharide (LPS) (nitrite concentration in medium 57 ⫾ 1 ␮M for resident macrophages stimulated with LPS versus 31 ⫾ 1 ␮M for resident macrophages stimulated with microsporidian spores and 36 ⫾ 4 ␮M for M. caulleryi prestimulated macrophages; P ⬍ 0.05). Extracellular release of reactive oxygen species (ROS) by resident macrophages in response to microsporidian spores was similar to that in response to Kluyveromyces lactis yeast cells and to that in response to phorbol myristate (a stimulator of protein C kinase). Intracellular ROS production by resident macrophages in response to microsporidian spores was similar to that produced in response to yeast cells. Both extracellular ROS production and intracellular ROS production (in response to all stimuli) were significantly lower after in vivo prestimulation of macrophages with microsporidian spores. These results demonstrate that microsporidian spores of species other than those that habitually infect mammals are capable of modulating the respiratory burst of rat peritoneal macrophages. Such modulation may contribute to avoidance by the microsporidian of cytotoxic responses associated with the respiratory burst. 䉷 2001 Academic Press Index Descriptors and Abbreviations: rat; microsporidian; respiratory burst; nitric oxide; macrophages.

INTRODUCTION In mammals, very little is known about cell-mediated immune responses to microsporidian parasites and about the mechanism by which microsporidians are able to evade the host immune system (Didier et al. 1994). Most such studies have focused on the role of macrophages in responses to microsporidians (Weidner and Sibley 1985; Didier and Shadduck 1994; Didier et al. 1994; Didier 1995; Khan and Moretto 1999). Certain species of microsporidians are capable of disseminating through the body using macrophages as vehicles (Canning and Lom 1986). The survival and replication of such species in macrophages are associated with an absence of phagosome–lysosome fusion (Weidner 1975; Schmidt and Shadduck 1984). Protozoan parasites ingested by macrophages are normally destroyed by the toxic activity of reactive oxygen and nitrogen species (ROS and RNS) generated by the respiratory burst response; survival within the macrophage thus indicates some sort of defense against this response (Maue¨l 1982, 1996). Axenic culture techniques have not been developed for microsporidians (Weiss and Vossbrinck 1998). Several species have been grown in tissue culture, but obtainment of spores in this way can take 3–6 weeks, and they must generally then be purified on a Percoll gradient (Shadduck 1969; Shadduck et al. 1979, 1990; Didier et al. 1991; Aldras et al. 1994; Furuya et al. 1995). In the present study we therefore used spores of Microgemma caulleryi, which are readily obtained in large quantities from the greater sand-eel (Hyperoplus lanceolatus) (Leiro et al. 1999).

To whom correspondence should be addressed. Fax: ⫹81-593316. E-mail: [email protected]. 1

0014-4894/01 $35.00 Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved.

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2 In addition, the use of M. caulleryi spores was of interest for two further reasons. First, previous studies of rodent cellmediated immune responses to microsporidians have used microsporidian species for which rodents are a natural host (Schmidt and Shadduck 1984; Hermanek et al. 1993; Didier and Shadduck 1994; Didier et al. 1994; Didier 1995; Achbarou et al. 1996; Khan and Moretto 1999): there have been no previous studies of responses to microsporidians naturally occurring in other hosts. Second, for many mammal-infecting microsporidians, we know neither the precise mode of transmission nor the natural reservoir (Kotler and Orenstein 1998), and it has been suggested that some humaninfecting microsporidians are transmitted to human via animals (Deplazes et al. 1996). Indeed, it has recently been suggested that AIDS patients may be infected with microsporidians perorally by ingestion of inadequately cooked fish (Cheney et al. 2000). In the present study, we thus investigated the respiratoryburst responses to viable M. caulleryi spores of rat resident peritoneal macrophages and of peritoneal macrophages from rats stimulated 5 days previously with such spores.

MATERIALS AND METHODS Experimental animals. Male Sprague–Dawley rats weighing 200– 300 g were obtained from Charles River Laboratory (France) and were provided with food and water ad libitum. Parasites and stimuli. Spores of the microsporidian M. caulleryi (Leiro et al. 1999) were isolated from xenomas (massively hypertrophic infected cells) in the liver of naturally infected greater sand-eels H. lanceolatus (Le Sauvage 1824) as described previously (Este´vez et al. 1992) and stored at 4⬚C in sterile seawater containing 0.1% sodium azide. Spore viability was confirmed by the method of Modha et al. (1997) as described previously (Leiro et al. 2000). Briefly, the spores were incubated in ethidium bromide (10 ␮g ml⫺1) for 15 min and then mounted for fluorescence microscopy without washing; damaged (nonviable) spores show red fluorescence. Kluyveromyces lactis (strain NRRL-41140) cells were cultured on NPD medium (Difco, USA) at 30⬚C at a growth rate of 0.07 h⫺1, washed with distilled water, and freeze-dried. A stock solution (2 mg/ml) of phorbol 12-myristate 13acetate (PMA) (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide and stored in the dark at ⫺80⬚C until use. A stock solution of lipopolysaccharide (LPS) from Escherichia coli serotype 0111:B4 (Sigma) was made up at 10 ␮g/ml in phenol-red-free Dulbecco’s modified Eagle’s medium (DMEM). Isolation of rat peritoneal-exudate macrophages. For induction of inflammatory responses, rats were injected intraperitoneally with 5 ml of PBS containing 109 M. caulleryi spores, and peritoneal exudate was extracted 5 days later. Rat resident and inflammatory peritoneal macrophages were obtained from euthanized rats by cervical dislocation, in a laminar flow chamber to ensure sterile conditions. The abdomen of the rat was wetted with 70% ethanol to achieve sterilization,

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a midline incision was then made with sterile scissors, and the abdominal skin was retracted. Thirty milliliters of Hanks’ balanced salt solution (HBSS) was then injected into the peritoneal cavity using a syringe with a 19-gauge needle. After gentle abdominal massage, about 30 ml of fluid was extracted using the same syringe and transferred to 50ml sterile polypropylene tubes on ice. A 20-␮l aliquot was then extracted for cell counting in a hemocytometer. The remaining cells were washed once by centrifugation at 400g for 10 min at 4⬚C and resuspended to a concentration of 1 ⫻ 106 cells/ml in HBSS. Aliquots (100 ␮l) of the cell suspension were added to the wells of 96-well microculture plates (Corning, USA) or placed on microscope slides and left for 90 min in a humidified incubator at 37⬚C, 5% CO2, to allow adhesion. Nonadherent cells were then removed by gently washing with HBSS. More than 97% of the remaining cells showed nonspecific esterase activity, determined as per Strober (1997), indicating that they were macrophages, and in what follows adherent cells isolated in this way are referred to as macrophages. Assay of nitrite production by stimulated macrophages. Macrophages that had been prestimulated in vivo with M. caulleryi spores were incubated with the same stimulus (100 spores or K. lactis cells per adherent cell, in HBSS), in all cases at 37⬚C with 5% CO2 for 90 min. After adhesion and phagocytosis, the wells were gently washed with HBSS and then incubated for 48 h with 100 ␮l of phenol-redfree DMEM containing 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, and 10 U/ml of recombinant murine interferon-␥ (IFN-␥, Genzyme, USA). In some experiments, the soluble stimulant Escherichia coli LPS (100 ng/ml), or the arginine analogue N-monomethylL-arginine monoacetate (L-NMMA, Calbiochem, San Diego, CA; 250 ␮M), was included in the medium. Nitrite production in the culture supernatants was assayed by the Griess reaction (Green et al. 1982): 100 ␮l of culture supernatant was added to 100 ␮l of Griess reagent (1% sulfanilamide, Sigma, and 0.1% napththylenediamine hydrochloride in 2.5% H3PO4) and then incubated for 10 min at room temperature, and absorbance was measured at 530 nm using an ELISA reader (Titertek Multiscan, Flow Laboratories, Finland). Nitrite concentration was calculated with reference to a standard curve obtained using NaNO2 (1–256 ␮M in culture medium). Assay of ROS production by stimulated macrophages. The production of ROS during the respiratory burst response of macrophages was investigated both extra- and intracellularly, using OxyBURST Green probes (Molecular Probes, The Netherlands) that emit fluorescence when they are oxidized by ROS produced during the respiratory burst (Ryan et al. 1990). For quantification of extracellular release of ROS, we used the OxyBURST Green H2HFF BSA reagent, i.e., bovine serum albumin coupled to dihydro-2⬘,4,4,6,7,7⬘-hexafluorofluorescein. A 1 mg/ml stock solution of the reagent was made up in HBSS containing 2 mM sodium azide and stored at 4⬚C in the dark. For the assay, macrophages were incubated with the reagent at 10 ␮g/ml in the wells of 96-well flat-bottom microtiter plates (Corning) for 2 min at 37⬚C and then stimulated by addition of 10 ␮g/ml of PMA. For negative controls, we added 100 ␮M L-(⫹)-ascorbic acid to the incubation medium. For quantification of intracellular production of ROS, we used the amine-reactive OxyBURST Green H2DCFDA, which must first be conjugated to the particles under study. To this end, 1 mg of the reagent was incubated with 10 mg of particles (microsporidian spores or yeast cells) in 0.05 M borate buffer, pH 8.3, in the dark for 60 min at room temperature with continuous gentle shaking. To hydrolyze the acetyl groups, we then added 0.1 ml of 1.5 M hydroxylamine, pH 8.5, to the reaction mixture and incubated it for 1 h at room temperature with stirring and then overnight at 4⬚C without stirring.

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Finally, the conjugate was separated from the unreacted reagent and hydroxylamine by multiple washes in which the substrate was pelleted by centrifugation at 5000g for 5 min, resuspended in HBSS, and stored in the dark at 4⬚C. For the assay, 1-␮l aliquots of the conjugated spore or cell suspension were added to the wells of 96-well microtiter plates containing macrophages (macrophage:spore/cell ratio 1:100). For negative controls, we added 1% sodium azide to the incubation medium. ROS release was quantified in a microplate fluorescence reader (BioTek Instruments, USA) on the basis of fluorescence release (excitation 488 nm, detection 530 nm) over a 45-min period. The rate of fluorescence release is proportional to the amount of ROS generated. Intracellular formation and extracellular release of ROS were expressed in terms of change fluorescence per unit time (arbitrary units per minute). Statistical analysis. The statistical significance of differences between means was determined (a) by unpaired two-tailed Student’s t tests when variances were equal (Fisher’s F test, P ⬎ 0.05) or (b) by Welch’s t test when variances were different. P values of less than 0.05 were considered significant.

RESULTS

RNS production by resident and microsporidian-prestimulated macrophages. We determined nitrite production by rat resident peritoneal macrophages and rat peritoneal macrophages prestimulated in vivo with spores of M. caulleryi, in response to stimulation in vitro with LPS, M. caulleryi spores, or K. lactis cells (Fig. 1). For both resident and microsporidian-prestimulated macrophages, nitrite production was significantly lower after stimulation with spores than after stimulation with LPS or yeast cells (Figs. 1A–1D). Nitrite production after stimulation with spores plus LPS was likewise relatively low (Fig. 1A). Basal nitrite production by microsporidian-prestimulated macrophages (i.e., nitrite production by prestimulated macrophages not stimulated in vitro) was significantly higher than basal nitrite production by resident macrophages (nitrite concentration in medium was 36 ⫾ 1 ␮M for prestimulated macrophages versus 14 ⫾ 1 ␮M for resident macrophages; P ⬍ 0.001) (Figs. 1C and 1D versus Figs. 1A and 1B). However, nitrite production by microsporidian-prestimulated macrophages in response to LPS (Fig. 1C) and yeast cells (Fig. 1D) did not differ significantly from nitrite production by resident macrophages in response to the same stimuli (Figs. 1A and 1B). Extracellular ROS production by resident and microsporidian-prestimulated macrophages. Extracellular ROS production by resident and microsporidian-prestimulated macrophages in response to stimulation in vitro with PMA, M. caulleryi spores, or K. lactis cells was investigated using

H2HFF-BSA (Fig. 2). For negative controls, the ROS-scavenger L-(⫹)-ascorbic acid was included in the medium. Extracellular ROS production by resident macrophages in responses to microsporidian spores (Fig. 2a) was not significantly higher than ROS production in response to PMA (Fig. 2A) and similar to that in response to yeast cells (Fig. 2B). Extracellular ROS production by microsporidianprestimulated macrophages in response to microsporidian spores was below the detection limits, regardless of the stimulus used (spores, yeast cells, or PMA), with the values obtained being similar to (Fig. 2D) or even lower than (Fig. 2C) those obtained without in vitro stimulation. The inclusion of L-(⫹)-ascorbic acid in the incubation medium significantly reduced net ROS production (Figs. 2A and 2B). Intracellular ROS production by resident and microsporidian-prestimulated macrophages. Intracellular ROS production by resident and microsporidian-prestimulated macrophages in response to stimulation in vitro with M. caulleryi spores or K. lactis cells was investigated using H2DCFDA conjugated to the stimulant particles (Fig. 3). For negative controls, sodium azide was included in the medium. Intracellular ROS production by resident macrophages in response to microsporidian spores did not differ significantly from that in response to yeast cells (Fig. 3A), while intracellular ROS production by microsporidian-prestimulated macrophages was significantly lower, in response to both microsporidian spores (mean increase in fluorescence was 470 ⫾ 85 units per minute for resident macrophages versus 202 ⫾ 39 units per minute for prestimulated macrophages; P ⬍ 0.05) and yeast cells (mean increase in fluorescence was 283 ⫾ 70 units per minute for resident macrophages versus 75 ⫾ 20 units per minute for prestimulated macrophages; P ⬍ 0.05) (Fig. 3B). The inclusion of sodium azide in the incubation medium significantly reduced net ROS production (Figs. 3A and 3B).

DISCUSSION The relationship between macrophage cytotoxicity and enhanced production of reactive nitrogen and oxygen intermediates in the killing of intracellular parasites is well known (Maue¨l 1982; Thorne and Blackwell 1983; Granger 1991; Liew 1993; Didier 1995; Clark and Rockett 1996; Piedrafita and Liew 1998). In macrophages, nitric oxide (NO) is generated by the enzymatic conversion of L-arginine to L-citrulline by inducible NO synthase (iNOs); full induction of NO in activated macrophages requires IFN-␥, tumor necrosis factor-␣ (TNF␣), or other cytokines or lipopolysaccharides

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FIG. 1. Nitrite levels in supernatants of 48-h cultures of rat resident peritoneal macrophages (A and B) and macrophages prestimulated in vivo with viable microsporidian spores (C and D) in response to in vitro exposure to Escherichia coli LPS (100 ng/ml), viable Microgemma caulleryi spores (A and C), Kluyveromyces lactis cells (B and D), or LPS plus viable spores or yeast cells, in the presence or absence of the iNOsinhibitor L-NMMA (250 ␮M). Bars show means ⫾ SE (n ⫽ 3 experiments, each using pooled macrophages from three rats). Asterisks indicate significant differences (*P ⬍ 0.05; **P ⬍ 0.01) with respect to LPS-stimulated cultures.

(Clark and Rockett 1996). In primary mouse peritoneal macrophages, LPS and IFN-␥ caused significant biosynthesis of nitrogen oxides only when the cells were prestimulated with thioglycollated broth or Bacillus Calmette-Gue´rin (Stuehr and Marletta 1987); in rats, however, resident peritoneal macrophages produce NO in response to relatively low doses of a single exogenous activating stimulus, including LPS (Lavnikova et al. 1993; Jorens et al. 1991). In fishes, for example, microsporidians induce much lower levels of RNS than “standard” stimuli such as LPS (Leiro et al. 2000). In the present study, the stimulation of rat peritoneal macrophages with microsporidian spores obtained from a fish generated significantly lower nitric oxide levels than stimulation of the same cells with lipopolysaccharide or yeasts. NO has

been shown to be an important immunoregulatory molecule in protozoan infections (Sternberg and McGuigan 1992; Rockett et al. 1994; Candolfi et al. 1995), including microsporidian infections (Khan and Moretto 1999). Specifically, NO regulates IL-2 and IFN-␥ production by TH1 cells (Taylor-Robinson et al. 1994). In experimental infections of IL-2/IFN-␥-deficient rats with the microsporidian Encephalitozoon cuniculi, it was demonstrated that both cytokines are necessary for protection against microsporidian infection (Khan and Moretto 1999), although the role of NO in this protection is controversial (Didier 1995; Khan and Moretto 1999). In the present study, we found that NO production by rat macrophages in response to joint stimulation with

MACROPHAGE RESPONSES TO MICROSPORIDIANS

FIG. 2. Extracellular ROS production (measured as the mean increase in fluorescence emission by oxidized OxyBURST Green H2HFF-BSA, in arbitrary units per minute) by rat resident peritoneal macrophages (A and B) and macrophages prestimulated in vivo with viable microsporidian spores (C and D) in response to in vitro exposure to 10 ␮g/ml of PMA or to viable Microgemma caulleryi spores (A and C), Kluyveromyces lactis cells (B and D) or PMA plus viable spores or yeast cells, in the presence or absence of the ROS scavenger L(⫹) ascorbic acid (100 ␮M). Values shown are means ⫾ SE (n ⫽ 3 experiments, each using pooled macrophages from three rats). Asterisks indicate significant difference (P ⬍ 0.05) with respect to group B.

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FIG. 3. Intracellular ROS production (measured as the mean increase in fluorescence emission by oxidized OxyBURST H2DCFDA succinimidyl ester, in arbitrary units per minute) by rat resident peritoneal macrophages (A) and macrophages prestimulated in vivo with viable microsporidian spores (B), in response to in vitro exposure to H2DCFDA-coupled Microgemma caulleryi spores or Kluyveromyces lactis cells, in the presence or absence of the ROS scavenger sodium azide (1%). Values shown are means ⫾ SE (n ⫽ 3 experiments each using pooled macrophages from three rats). Asterisks indicate significant differences (P ⬍ 0.05) between groups B and C or groups D and E.

microsporidian spores and LPS was lower than that in response to stimulation with LPS alone. Some studies have investigated the interactive effects of different stimuli on NO production, and it has been observed that preexposure of peritoneal macrophages to low concentrations of LPS also suppresses the subsequent induction of iNOs by IFN␥ (Bogdan et al. 1993). Furthermore, macrophages from rats stimulated in vivo with microsporidian spores are capable

of NO production; however, the NO response of these cells to microsporidian spores is markedly lower than their NO response to yeast cells or LPS. Phagocytosis itself may increase iNOs activity: for example, phagocytosis of Leishmania promastigotes or latex beads by murine macrophages enhances IFN-␥-stimulated nitrite production, and could be an important mechanism for up-regulating their microbicidal activity (Corradin et al. 1991). Ingestion of zymosan, but

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not of latex beads or silica, also acts synergistically with LPS to increase iNOs activity (Cunha et al. 1993). There are various possible explanations for the scant NO production by rat macrophages prestimulated in vivo with microsporidian spores. These explanations include (a) stimulation of inhibitory cytokines, including IL-4, IL-10, and IL-13 (Jorens et al. 1995); (b) inhibition of the production of IFN-␥ or TNF-␣ (both of which stimulate NO production; Langermans et al. 1992); or (c) production of prostaglandins and phosphodiesterase inhibitor, inducing prolonged elevation of intracellular cyclic AMP levels and possibly causing marked reduction of NO production by macrophages (Bulut et al. 1993). NO can combine with superoxide anion to form the potent oxidizing agent peroxynitrite (ONOO⫺), and thus energy metabolism in macrophages may be determined to a significant extent by iNOs products (Jorens et al. 1995; DarleyUsmar et al. 1995). In macrophages, activation of the respiratory burst correlates with translocation of protein kinase C (PKC) from the cytosolic fraction to the cell membrane fraction (Myers et al. 1985) and is accompanied by PKCdependent phosphorylation of specific cellular proteins, including components of NADPH oxidase (Nauseef et al. 1991; Hampton and Winterbourn 1995). Among the agonists of the PKC-induced burst is the phorbol ester PMA, a fast activator that is widely used to trigger the production of O2⫺ (Bjo¨rquist et al. 1994). In the present study, microsporidians clearly reduced both intra- and extracellular ROS production by rat macrophages: specifically, ROS production (whether in response to microsporidian spores, yeast cells, or PMA) by macrophages that had been prestimulated in vivo with microsporidian spores was markedly lower than that by normal resident macrophages. This inhibitory effect is particularly noteworthy if we bear in mind that in vivo prestimulation typically enhances the macrophage respiratory burst: for example, mouse peritoneal macrophages produce significant levels of superoxide in response to PMA only after in vivo prestimulation with BCG or, less effectively, with thioglycollate or lipopolysaccharide (Johnston et al. 1978). Other intracellular parasites likewise interfere with the macrophage respiratory burst: for example, Leishmania interferes with PKC-dependent signal transduction pathways (Reiner 1994), and Toxoplasma gondii induces only low levels of oxidative activity (Wilson et al. 1980). Other mechanisms might also be proposed to explain the observed blockage of the rat macrophage respiratory burst by microsporidians: for example, phorbol esters capacitate macrophage complement receptors for phagoctyosis (Wright and Silverstein 1982) and induce phosphorylation of complement receptor 1 (Changelian and Fearon 1986) and the ␤ subunit of complement receptor 3 (Buyon et al. 1990), promoting activation of

phagocytosis. In studies with turbot macrophages, we have observed that complement does not opsonize the phagocytized spores, and it has been proposed that phagocytosis of microsporidians is independent of complement and might be related to the binding of parasites to macrophage-surface lectins (Leiro et al. 1996), as has been reported in the phagocytosis of yeast cells (Sharon 1984). That the parasite is not efficiently opsonized might indicate modulation of the functional properties of macrophage CR3, maintaining it in an inactive state that prevents efficient phagocytosis and blocks activation of the respiratory burst. Finally, we found that 100 ␮M L-(⫹)-ascorbic acid was an effective scavenger of ROS released extracellularly in response to all the stimuli tested. In studies of human neutrophils stimulated with PMA, O2⫺ was likewise efficiently scavenged with 100 ␮M ascorbic acid (Bjo¨rquist et al. 1994). The single oxygen and hydrogen peroxide scavenger sodium azide was likewise a potent inhibitor of intracellular ROS production: at a concentration of 150 mM, this compound completely inhibited net intracellular ROS release in response to both microsporidian spores and yeast cells. Sodium azide likewise effectively inhibited net ROS release by human neutrophils stimulated with zymosan or PMA (Hasegawa et al. 1997). In conclusion, the results of the present study indicate that, by comparison with other stimuli, viable spores of the microsporidian M. caulleryi only slightly stimulate the respiratory burst of rat resident peritoneal-exudate macrophages. In addition, experiments involving prior stimulation of macrophages in vivo, by injection of viable M. caulleryi microsporidian spores into the peritoneal cavity, indicate that these microsporidians in fact actively inhibit the respiratory burst response of rat macrophages, by a mechanism as yet unknown.

ACKNOWLEDGMENTS The authors thank Dr. M. I. G. Siso of the Biochemical and Molecular Biology Laboratory (Faculty of Sciences, University of A Corun˜a, Spain) for providing the Klyuveromyces lactis yeast cells used in this study. This work was financially supported by Grant PGIDT99MAR20301 from the Xunta de Galicia and Grant 1F097/0032 from the CYCIT (Ministerio de Educacio´n y Cultura) of Spain.

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