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Lipopolysaccharide-mediated enhancement of zymosan phagocytosis by RAW 264.7 macrophages is independent of opsonins, laminarin, mannan, and complement receptor 3 Ana-Lucı´a Fuentes, PhD,a Leonard Millis, PhD,b Jacqueline Vapenik, MN,c and Lynette Sigola, MBChB, PhDb,* a
Department of Natural Sciences, LaGuardia Community College, City University of New York, Long Island City, New York b Biology Department, Faculty of Science and Technology, New Westminster British Columbia, Canada c Nursing Department, Faculty of Health Sciences, Douglas College, New Westminster British Columbia, Canada
article info
abstract
Article history:
Background: Fungal and bacterial coinfections are common in surgical settings; however,
Received 10 August 2013
little is known about the effects of polymicrobial interactions on the cellular mechanisms
Received in revised form
involved in innate immune recognition and phagocytosis.
27 November 2013
Materials and methods: Zymosan particles, cell wall derivatives of the yeast Saccharomyces
Accepted 11 March 2014
cerevisiae, are used to model fungal interactions with host immune cells since they display
Available online 17 March 2014
carbohydrates, including beta-glucan, that are characteristic of fungal pathogens. Using in vitro cell culture, RAW 264.7 macrophages were challenged with zymosan, and phago-
Keywords:
cytosis determined via light microscopy. The effects of different concentrations of lipo-
Lipopolysaccharide (LPS)
polysaccharide (LPS) on zymosan phagocytosis were assessed. In addition, the transfer of
Macrophage Phagocytosis
supernatant from LPS-treated cells to naı¨ve cells, the effects of soluble carbohydrates laminarin, mannan, or galactomannan, and the impact of complement receptor 3 (CR3)
Zymosan
inhibition on phagocytosis were also determined.
Beta-glucans
Results: LPS enhanced phagocytosis of zymosan in a dose-dependent manner. Transfer of
CR3
supernatants from LPS-primed cells to naı¨ve cells had no effect on phagocytosis. Laminarin
Laminarin
inhibited zymosan phagocytosis in naı¨ve cells but not in LPS-primed cells. Neither mannan,
Opsonin
galactomannan, nor CR3 inhibition had a significant effect on ingestion of unopsonized
RAW 264.7
zymosan in naı¨ve or LPS-treated cells. Conclusions: Zymosan recognition by naı¨ve cells is inhibited by laminarin, but not mannan, galactomannan, or CR3 inhibition. LPS enhancement of phagocytosis is laminarin insensitive and not mediated by supernatant factors or zymosan engagement by the mannose or CR3 receptors. Our data suggest alternative mechanisms of zymosan recognition in the presence and absence of LPS. ª 2014 Elsevier Inc. All rights reserved.
* Corresponding author. Biology Department, Douglas College, P.O. Box 2503, New Westminster, B.C., Canada V3L 5B2. Tel.: þ1 604 527 5233; fax: þ1 604 527 5095. E-mail address: [email protected] (L. Sigola). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.03.024
j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 9 ( 2 0 1 4 ) 3 0 4 e3 1 2
1.
Introduction
Fungal infections may cause minor illnesses involving superficial structures of the mucosae or integumentary system and more severe disease with infiltration of deep-seated tissues and/or systemic invasion. Severe fungal infections are more prevalent in patients immunocompromised by human immunodeficiency virus, patients with malignancies on antineoplastic chemotherapy, and transplant patients on immunosuppressants [1,2]. Infections with fungal pathogens, particularly Candida albicans and Candida glabrata, have increased in frequency in intensive care units [3] and commonly occur concomitantly with bacterial pathogens [4]. Furthermore, increased mortality rates due to postsurgical fungal sepsis correlate with the use of multiple antibiotics, prolonged use of antibiotics, and with concomitant bacterial sepsis [5], observations which suggest the importance of understanding immune responses to fungi in the context of simultaneous bacterial infection. Endotoxin is associated with enhanced proinflammatory mediator secretion and a cascade of reactions that often results in death [6]. In some situations, however, endotoxin exposure has been reported to augment antifungal resistance in the host. For example, when given before either inoculation with Aspergillus fumigatus or Cryptococcus neoformans, lipopolysaccharide (LPS) significantly reduced mortality and lowered fungal tissue burdens in mice [7,8]. Similar studies reported reduced C glabrata burdens in the tissues of mice exposed to either Escherichia coli or the bacterium’s LPS, compared with control mice [9]. Of additional note in this latter study, was the observation of enhanced C glabrata phagocytosis by macrophages exposed to LPS in vitro. C albicans phagocytosis is also increased in freshly isolated human peripheral blood monocytes after stimulation with LPS [10]. Significant progress has been made in understanding the host immune response to fungal pathogens. Macrophages contribute to fungal resistance through their role as professional phagocytes by seeking out, ingesting, and killing microbes; they also secrete chemokines and cytokines, release microbicides including nitric oxide and reactive oxygen intermediates, and activate adaptive T cell immunity after fungal component antigen processing and presentation [11e13]. Macrophage recognition of pathogens and their products is achieved by host expression of germline-encoded pattern recognition receptors (PRRs); these are soluble or membrane-bound receptors which recognize microorganisms at specific conserved targets called pathogen-associated molecular patterns (PAMPs) [14]. Beta-glucans are carbohydrates found in fungi, plants, and some bacteria [15]. In fungi, they serve as significant PAMPs for several species including C albicans [16], A fumigatus [17], Pneumocystis carinii [18], Coccidioides posadasii [19], S cerevisiae [20], and its cell wall derivative, zymosan. Zymosan is frequently used to model fungal recognition and phagocytosis by macrophages because it is composed of large amounts of beta-glucan, in addition to small quantities of mannose and chitin [21]. We have previously reported that laminarin, a soluble low molecular weight beta-glucan, has differential effects on zymosan phagocytosis in the absence and presence of LPS
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[22]. Laminarin inhibited zymosan phagocytosis in the absence of LPS, consistent with beta-glucan serving as an important PAMP during macrophage recognition and phagocytosis of zymosan. The presence of LPS significantly enhanced zymosan phagocytosis, and laminarin failed to inhibit zymosan ingestion in cells primed with LPS, suggesting the presence of laminarin sensitive and insensitive mechanisms for macrophage recognition of zymosan in the absence and presence of LPS, respectively. In this study, we investigated the potential roles that any supernatant factors such as opsonins, the mannose receptor, or the complement receptor 3 (CR3) may play in contributing to the enhanced zymosan phagocytosis induced by LPS.
2.
Materials and methods
2.1.
Reagents
Zymosan prepared from S cerevisiae, laminarin from Laminaria digitata, mannan from S cerevisiae, galactomannan from the locust bean gum of Ceratonia siliqua, and LPS from E coli serotype 055:B5 were all obtained from Sigma-Aldrich (St. Louis, MO). To assess the role of the CR3 receptor in phagocytosis experiments, functional gradeepurified anti-mouse CD11b antibody M1/70 (Integrin aM or Mac-1a) and its isotype-matched rat antimouse IgG2b control antibody were obtained from eBioscience (San Diego, CA). For phagocytosis assays, macrophages were cultured in sterile tissue culture medium composed of Roswell Park Memorial Institute 1640 medium (Life Technologies, Burlington, ON), supplemented with 10% heat-inactivated fetal calf serum (Life Technologies), 100 U/mL penicillin (Life Technologies), 100 mg/mL streptomycin (Life Technologies), 25 mM N-2-hydroxyethylpiperazine-NV-2-ethanesulphonic acid (Life Technologies), and 10 mM L-glutamine (Life Technologies; R10). Dulbecco Modified Eagles Medium (DMEM) (Life Technologies) was used for the growth of RAW 264.7 macrophages and supplemented in the same way as the Roswell Park Memorial Institute to form D10. Sterile plasticware, noncharged, was obtained from Corning Inc (Fisher Scientific, Toronto, ON), and used for all experiments.
2.2.
Macrophage cell culture
RAW 264.7 macrophages from American Tissue Type Culture Collection were obtained from Cedarlane Laboratories, Burlington, ON. Cells were maintained in D10 and passaged every 3 d when >90% confluent. In preparation for phagocytosis assays, cells were harvested and centrifuged at room temperature for 7 min at 1100 rpm, resuspended in R10, counted, and viability assessed using trypan blue dye (Sigma-Aldrich, St. Louis, MO) exclusion. Viability in all experiments was >90%.
2.3.
Opsonization of zymosan
Serum opsonization of zymosan was achieved by incubating 1 108 zymosan particles per ml suspended in R10 with an equal volume of fetal calf serum at 37 C for 30 min with
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frequent shaking. The opsonized zymosan particles were then washed four times in R10 before use in phagocytosis assays.
2.4.
Macrophage phagocytosis assay
For phagocytosis assays, 1 105 RAW 264.7 macrophages in R10 medium were plated onto 12-mm glass coverslips (Fisher Scientific, Toronto, ON) placed in sterile 24-well tissue culture plates (Costar, Fisher Scientific, Toronto, ON), and macrophages allowed to adhere in a humidified 5% CO2 incubator for 30 min at 37 C. Zymosan particles were added to the cells at a zymosan to macrophage ratio of 10:1. Phagocytosis was allowed to proceed for 1 h at 37 C in a humidified 5% CO2 incubator. In experiments where the effects of either carbohydrates or anti-CD11b were assessed, cells were incubated in the presence of these reagents for 15 min before addition of zymosan. Phagocytosis was terminated by vigorously washing off noningested particles with warm R10 three times. Glass coverslips were air-dried for 15 min, stained using Wright stain (Sigma-Aldrich, St. Louis, MO) and then washed with water, then coverslips air-dried and mounted onto glass microscope slides using Permount (Fisher Scientific, Toronto, ON). Slides were examined by light microscopy at 400 or 600 magnification using a Nikon Eclipse 50i photomicroscope (Nikon Canada Inc, Richmond, BC). Phagocytosis was determined from photomicrographs at 400 or 600 magnification by counting at least 100 macrophages in at least four different fields of view for each experimental treatment. Phagocytosis was assessed as percentage of phagocytic cells (PP), mean number of particles per cell (MNP) and as phagocytic index (PI) determined as PI ¼ PP MNP. Photomicrographs of cells for illustrations were taken at 1000 magnification.
2.5.
Statistical analysis
Results are expressed as the mean standard error of the mean. Statistical analysis was performed using the VassarStat online program using either the unpaired two-tailed Student t-test or analysis of variance followed by the post hoc Tukey test, as appropriate. A P value of 0.05 was considered statistically significant. All experiments were repeated at least twice with similar results.
3.
Results
3.1. LPS enhances zymosan phagocytosis in a concentration-dependent manner To investigate the effect of LPS on zymosan phagocytosis, we compared ingestion of macrophages cultured with medium alone versus cells cultured with three different concentrations of LPS at 1, 10, and 100 ng/mL, respectively (Fig. 1). LPS increased all measures of phagocytosis, and its effects on MNP and PI were statistically significant (P ¼ 0.002 and P ¼ 0.0006, respectively). LPS treatment enhanced the efficiency of phagocytosis as reflected by an increase in MNP from 4.5 1.1 for cells cultured in medium alone to 17.5 3 for cells cultured with LPS 100 ng/mL (Fig. 1B). PI also
Fig. 1 e LPS enhances zymosan phagocytosis in a concentration-dependent manner. Cells (1 3 105) were cultured in either medium alone or LPS at 1, 10, and 100 ng/mL respectively, for 16 h before addition of zymosan particles at a macrophage-to-zymosan ratio of 1:10. Phagocytosis was allowed to proceed for 1 h at 37 C in a 5% CO2 incubator, after which phagocytosis was terminated, cells stained and phagocytosis determined by light microscopy and expressed as percentage phagocytosis (A), mean number of particles ingested per cell (B) and PI (C). The percentage of cells in either medium alone or LPS, ingesting 0e5, 6e10, 11e15, and >16 zymosan particles per cell was also determined (D). Photomicroscopy was performed under oil immersion at 31000 magnification (E) and shows representative cells cultured in either medium alone or specified concentrations of LPS for 16 h, and then cells presented with zymosan particles for 1 h. Scale bar on photomicrograph represents 10 mm. Results presented are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01 for specified samples versus medium alone.
increased significantly with LPS pretreatment. We also compared the effect of LPS on the percentage of cells ingesting various quantities of zymosan particles (Fig. 1D). Most (73%) of the naı¨ve cells cultured in medium alone
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Fig. 1 e (continued).
ingested 16 particles each. In contrast, treatment with LPS at 1, 10, and 100 ng/mL increased the percentage of cells with more than 16 zymosan particles each to 13%, 39%, and 53%, respectively. There was a progressive rise in the number of ingested particles within the cytoplasm of cells primed with increasing LPS concentrations, compared with naı¨ve cells cultured in medium alone (see photomicrographs in Fig. 1E). Together, these results suggest an increase in overall zymosan phagocytosis effected, in part, by augmentation of macrophage phagocytic avidity in association with rising LPS concentrations in cell cultures.
3.2. Supernatant transfer from LPS-stimulated cells to naı¨ve cells has little effect on zymosan phagocytosis We considered the possibility that supernatants from LPSprimed macrophages may contain cell-derived opsonins that contribute to the enhanced avidity for zymosan by cells pretreated with LPS for 16 h. To investigate this possibility, culture supernatants from cells primed with LPS for 16 h were removed and added to naı¨ve cells, and the effect of this transfer on phagocytosis compared with ingestion in cells cultured in either medium alone, with LPS for 16 h and supernatant retained, with LPS-stimulated cells from which the supernatant was washed off, and cells pretreated with LPS for only 20 min (see Fig. 2). Cells cultured for 16 h with LPS (0.5 ng/mL) showed significantly enhanced zymosan phagocytosis compared with cells cultured in medium alone (MNP for medium alone: 1.1 0.5 versus LPS 10.9 1.4). For cells cultured with LPS for 16 h from which the supernatants were washed off before zymosan addition, MNP was 8.6 0.1; although reduced when compared with cells primed for 16 h with LPS, this difference in MNP was not
statistically significant. For naı¨ve cells treated for 20 min with culture supernatants transferred from other cells primed with LPS for 16 h, MNP was 2.4 0.5, and thus similar to MNP observed in cells cultured in medium alone. Supernatant transfer from LPS 16 h cultures to naı¨ve cells also had little effect on PP or PI.
3.3. Laminarin, mannan, and galactomannan have little effects on LPS-induced augmentation of zymosan phagocytosis The surface of zymosan is rich in carbohydrates, particularly beta-glucan and mannose [21]. To assess the importance of carbohydrate recognition for zymosan phagocytosis, RAW 264.7 cells were exposed to zymosan in the presence or absence of soluble carbohydrates, specifically laminarin (a beta-glucan), mannan (a mannose receptor blocker), and galactomannan (a carbohydrate not present on zymosan; see Fig. 3A). Carbohydrates were added to naı¨ve cell cultures 15 min before addition of zymosan. Cells cultured in medium alone ingested zymosan with MNP of 3.7 1.2. The presence of mannan or galactomannan had little effect on phagocytosis (MNP 5.8 0.9 and 5.1 1.4, respectively). In contrast, laminarin suppressed phagocytosis of zymosan significantly, inhibiting MNP by 76% (MNP 0.9 0.5). Percentage phagocytosis and PI were similar in cells cultured in medium alone and with mannan or galactomannan, respectively; in contrast, laminarin significantly suppressed both percentage phagocytosis and PI (data not shown). We also examined the effects of these carbohydrates on the LPS-induced increase in phagocytosis (Fig. 3B). LPS significantly enhanced MNP (3.3 0.3 for medium alone versus 14.6 0.8 for LPS), and the presence of soluble carbohydrates in cell cultures did not ameliorate LPS modulation of zymosan ingestion.
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Fig. 2 e Supernatant transfer from LPS-primed cells to naı¨ve cells does not augment mean number of zymosan particles ingested. Cells (1 3 105) were cultured in either medium alone, with LPS 0.5 ng/mL for 16 h, with LPS for 16 h and supernatants (sups) washed off, with naı¨ve cells treated with supernatants from cells cultured with LPS for 16 h, or with LPS for 20 min before addition of zymosan particles at a macrophage-to-zymosan ratio of 1:10. Phagocytosis was allowed to proceed for 1 h at 37 C in a 5% CO2 incubator, after which phagocytosis was terminated, cells stained and viewed by light microscopy. Counts were performed and phagocytosis determined as percentage phagocytosis (A), mean number of particles ingested per cell (B) and PI (C). Results presented are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.0001, respectively, for specified samples versus medium alone.
Fig. 3 e Laminarin, mannan, and galactomannan have little effect on LPS-induced augmentation of zymosan phagocytosis. Cells (1 3 105) were cultured in either medium alone (A) or with LPS 1 ng/mL for 16 h (B), where indicated either laminarin, mannan, or galactomannan, respectively, each at 100 mg/mL, was added to cell cultures for 15 min before addition of zymosan particles at a macrophage-to-zymosan ratio of 1:10. Phagocytosis was allowed to proceed for 1 h at 37 C in a 5% CO2 incubator, after which phagocytosis was terminated, cells stained and phagocytosis determined by light microscopy and expressed as mean number of particles ingested per cell. Results presented are expressed as mean ± standard error of the mean. *P < 0.05 and **P < 0.01, respectively, for specified samples versus medium alone.
3.4. CR3 inhibition impairs phagocytosis of serumopsonized zymosan but has little impact on phagocytosis of unopsonized zymosan in the absence or presence of LPS The CR3 receptor has been reported to bind zymosan nonopsonically and mediate its recognition and subsequent phagocytosis [23]. Therefore, we examined the effect of specific CR3 blockade on zymosan ingestion in naı¨ve RAW cells and naı¨ve cells presented with serum-opsonized particles (see Fig. 4). Specific CR3 blockade with the anti-CD11b antibody
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M1/70 had little effect on ingestion of unopsonized zymosan by naı¨ve RAW cells, and this was similar for its isotypematched control antibody (MNP for medium alone: 2.2 0.2, M1/70: 2.2 0.2, and control antibody: 2.2 0.3). Similarly, CR3 inhibition had little effect on percentage phagocytosis and PI for unopsonized zymosan. Opsonization of zymosan with serum significantly enhanced phagocytosis by 132% (see Fig. 4B; MNP for unopsonized zymosan: 2.2 0.2 versus opsonized zymosan: 5.1 0.6). M1/70 significantly inhibited phagocytosis of opsonized zymosan by 35% (MNP 3.3 0.4), but the control antibody had little effect on MNP (opsonized zymosan: 5.1 0.6 versus control antibody plus opsonized zymosan: 6.5 1.1). CR3 inhibition also significantly suppressed PI for serum-opsonized zymosan. Finally, we compared the effects of CR3 inhibition, laminarin, and mannan on zymosan phagocytosis in naı¨ve cells and LPS-primed cells. As observed previously, naı¨ve cell ingestion of zymosan was significantly inhibited by laminarin (MNP for medium alone 4.3 0.8, MNP for laminarin 0.5 0.5), but mannan, CR3 inhibition, and the control antibody had little effect (3.1 0.7, 4 0.7, and 2.6 0.5, respectively; see Fig. 5A). We also studied the effect of CR3 receptor blockade on enhanced phagocytosis observed for unopsonized zymosan by macrophages primed for 16 h with LPS (see Fig. 5B). LPS significantly augmented zymosan ingestion (MNP for medium alone 4.3 0.8, MNP for LPS 9.9 0.5). The anti-CR3 and control antibodies had little effects respectively on the increased zymosan ingestion due to LPS (MNP for LPS plus M1/ 70: 12.4 2.8, MNP for LPS plus control antibody: 8.6 0.7), nor, as observed previously, was the LPS effect ameliorated by laminarin or mannan, which were used at higher concentrations of 400 mg/mL, respectively, in this experiment.
4.
Fig. 4 e CR3 inhibition impairs phagocytosis of serumopsonized zymosan but has little impact on phagocytosis of unopsonized zymosan. Cells (1 3 105) were cultured in either medium alone or with 20 mg/mL anti-CR3 antibody M1/70 or its isotype-matched control antibody (Ab) for 15 min at 4 C before addition of either unopsonized zymosan (dark bars) or serum-opsonized zymosan (light bars) at a macrophage-to-zymosan ratio of 1:10. Phagocytosis was allowed to proceed for 1 h at 37 C in a 5% CO2 incubator, after which phagocytosis was terminated, cells stained and phagocytosis determined by light microscopy and expressed as percentage phagocytosis (A), mean number of particles ingested per cell (B) and PI (C). Results presented are expressed as mean ± standard error mean. *P < 0.05 and ***P < 0.001, respectively.
Discussion
In this study we demonstrated that exposure of RAW 264.7 macrophages to LPS significantly enhanced the avidity and efficiency of zymosan phagocytosis in a concentrationdependent manner. Similar dose-dependent effects of LPS have been reported for human peripheral blood monocytes’ ingestion of C albicans [10] and increased phagocytic efficiency noted for RAW 264.7 cell uptake of E coli in macrophages exposed to lipid A [24]. We also confirmed that macrophage recognition of zymosan was laminarin sensitive in the absence of LPS but laminarin insensitive for LPS-primed cells [22]. Zymosan particles are composed primarily of betaglucan, and cell surface macrophage PRRs reported to bind to this carbohydrate include Dectin-1 [25], CR3 [26], SIGNR1 [27], scavenger receptors [28], and lactosylceramide [29]. The mannose receptor has also been implicated as a PRR for zymosan via recognition of mannan on these particles [30]. We investigated three possibilities that could account for increased phagocytosis in LPS-stimulated cells. First, the potential contribution of substances, such as opsonins, present in the supernatants of cells primed with LPS; second, the role of mannose receptors; and finally, we examined possible involvement of CR3.
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Fig. 5 e CR3 inhibition has little effect on phagocytosis of unopsonized zymosan in the presence of LPS. Cells were cultured in either medium alone (A) or pretreated with LPS at 1 ng/mL for 16 h (B). Fifteen minutes before the addition of zymosan particles at a macrophage-to-zymosan ratio of 1:10, cells were cultured with either laminarin (400 mg/mL), mannan (400 mg/mL), anti-CR3 antibody M1/70 (100 mg/mL) or its isotype-matched control antibody (100 mg/mL), and antibody pretreatments performed at 4 C. Phagocytosis was allowed to proceed for 1 h at 37 C in a 5% CO2 incubator, after which phagocytosis was terminated, cells stained and phagocytosis determined by light microscopy and expressed as mean number of particles ingested per cell. Results presented are expressed as mean ± standard error of the mean. **P < 0.01 for specified samples versus medium alone.
Zymosan binds soluble proteins, which may act as opsonins and these include Pentraxin 3 (PTX3) [31], C-reactive protein and serum amyloid protein [32]. Opsonins bind PAMPs on microorganisms, thus facilitating the recognition of numerous and varied microbes with the use of only a limited number of host receptors. Murine macrophages make very small quantities of C-reactive protein [33]; however they produce large amounts of PTX3 and serum amyloid protein in response to LPS stimulation [34,35]. Zymosan phagocytosis is enhanced in macrophages from PTX3 transgenic mice [31] whereas PTX3 binding to its targets is blocked by galactomannan [36]. In our experiments supernatant transfer from LPS-primed cells to naı¨ve cells had little effect on phagocytosis, and galactomannan had no effect on zymosan ingestion in neither naı¨ve nor LPS-stimulated cells.
Furthermore, the addition of exogenous PTX3 failed to increase zymosan uptake by cells (data not shown), suggesting little role for PTX3 during phagocytosis in our model. We noted a small decrease in MNP in cells primed overnight with LPS and where culture supernatants were removed, compared with cells where the supernatants were retained, but this reduction was not statistically significant. We speculate that LPS enhances the expression of particular cell surface receptors for zymosan and that opsonins play little role in LPSmediated augmentation of zymosan phagocytosis. Laminarin inhibited zymosan phagocytosis in cells cultured in medium alone, consistent with several reports on the significance of beta-glucans as PAMPs recognized by murine macrophages [37e39]. In the presence of LPS, however, we confirmed our previous finding that laminarin was ineffective in abrogating enhanced zymosan phagocytosis. Therefore, we examined the role of the mannose receptor in the LPSmediated increase in phagocytosis. Mannan binds mannose receptors [40] and SIGNR1 [41] and both PRRs contribute to zymosan recognition and phagocytosis. RAW cells lack SIGNR1 [42], and therefore this receptor likely does not explain the increase in zymosan phagocytosis due to LPS. In our experiments, mannan had little effect on ingestion of zymosan in cells cultured in the absence or presence of LPS, even when mannan concentrations as high as 1 mg/mL were used in cell cultures (data not shown). Li et al. [43] also showed that mannan had no effect on zymosan ingestion by murine resident peritoneal macrophages. CR3 also binds beta-glucans, and there is evidence in human neutrophils and macrophages [44], in addition to murine bone marrow derived macrophages [45], that CR3 is the primary PRR for this carbohydrate. Nonopsonic binding of zymosan to CR3 occurs via the lectin-binding site on CD11b [26]. Using the antiCD11b antibody M1/70, we abrogated the increase in phagocytosis of serum-opsonized zymosan over that of unopsonized particles, however M1/70 had no effect on either naı¨ve cell ingestion of unopsonized zymosan or LPS-induced augmentation of phagocytosis of unopsonized zymosan. This suggests little role for CR3 as a significant receptor for increased zymosan ingestion in cells pretreated with LPS. Our study suggests that RAW macrophages recognize betaglucan bearing particles via different mechanisms in the absence and presence of LPS. Similar to our observations with LPS, the toll-like receptor 9 ligand CpG enhanced phagocytosis of zymosan and C albicans, respectively [46], and the authors showed laminarin sensitive zymosan ingestion in naı¨ve cells, whereas cell primed with CpG increased zymosan intake but this was not ameliorated by laminarin. Much attention has focused on Dectin-1 as a zymosan receptor since Brown et al. [25] identified this C-type lectin (inhibited by laminarin) as the primary murine PRR for beta-glucans, although its expression is variable depending on cell type and maturation state [47]. In our study, we used RAW macrophages, which express little Dectin-1 [25]; furthermore, LPS has been reported to downregulate Dectin-1 expression in macrophages [48]. Based on these reports and our own observations, it is unlikely that Dectin-1 plays a major role in mediating the LPS effect on zymosan ingestion in RAW macrophages. However, in contrast to our findings and other studies, it is interesting to note that LPS-treated human monocytes have recently been
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reported to increase Dectin-1 transcription and cell surface expression with associated enhancement of C albicans phagocytosis [10]. Therefore, it seems possible that cell type and method of cell maturation may contribute to differences observed for LPS effects on Dectin-1 expression. Based on our data, it is unlikely that supernatants of LPSstimulated cells, the mannose receptor or CR3 recognition of zymosan, explain the enhancement of phagocytosis by LPS. Further investigation is required to examine what roles other receptors, including scavenger receptors and lactosylceramide, may play as alternative mechanisms of zymosan ingestion in cells previously exposed to LPS. Scavenger receptors are promiscuous transmembrane proteins expressed on a variety of cells, including macrophages, and they bind to a wide variety of targets such as apoptotic cells, microbes, and zymosan [28,49,50]. It is interesting to note that LPSmediated enhancement of C glabrata phagocytosis is associated with augmented expression of macrophage receptor with collagenous structure, a scavenger receptor recognized as a marker of macrophage activation [9]. Additional studies are required to determine what functional role macrophage receptor with collagenous structure, or other classes of scavenger receptors, may play in zymosan recognition and phagocytosis after exposure of cells to LPS. Lactosylceramide is a glycosphingolipid that contributes to the formation of plasma membrane lipid rafts and acts as a PRR that binds microbes, including C albicans [51]. It is predominantly expressed on neutrophil membranes and contributes to increased avidity of CR3 binding to nonopsonized zymosan [52]. Additional studies are required to examine any possible roles for this glycosphingolipid in LPS-mediated augmentation of zymosan ingestion by macrophages. The challenges of understanding innate immunity in the context of multiple interactions involving the host with commensal, symbiotic, and pathogenic microbes, in addition to intermicrobial cross talk, are intimately connected with recognizing the critical dynamic equilibrium that determines health and disease. There is mounting evidence for the influence of intestinal commensal bacteria in shaping innate immunity and for the importance of PRRs in the recognition of the microbiota for promotion of host-microbial symbioses [53,54]. These mutualistic interactions are not limited to bacteria and hosts, as there is also recent evidence for the importance of gut fungi in modulating bowel inflammation [55] and allergic reactions [56]. In the light of growing evidence of the importance of relationships between the immune system and microbiota in contributing to human health and disease, understanding the mechanisms of these interactions is critical for novel preventative and therapeutic approaches, especially when considering the growing prevalence of antimicrobial resistance and concomitant polymicrobial infections.
Acknowledgment This study was supported by the Douglas College Research and Scholarly Activity Fund. The work presented was carried out with the collaboration of all authors. A-.L.F. and L.S. designed the experiments, conducted the phagocytosis studies, and analyzed and
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interpreted the data. L.M. and J.V. maintained the cell line, stained slides, collected and analyzed the data, and contributed to discussions on data interpretation. All authors contributed to drafting the manuscript. A-.L.F. and L.S. critically reviewed and revised the manuscript.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
references
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Available online at www.sciencedirect.com
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Lipopolysaccharide-mediated enhancement of zymosan phagocytosis by RAW 264.7 macrophages is independent of opsonins, laminarin, mannan, and complement receptor 3 Ana-Lucı´a Fuentes, PhD,a Leonard Millis, PhD,b Jacqueline Vapenik, MN,c and Lynette Sigola, MBChB, PhDb,* a
Department of Natural Sciences, LaGuardia Community College, City University of New York, Long Island City, New York b Biology Department, Faculty of Science and Technology, New Westminster British Columbia, Canada c Nursing Department, Faculty of Health Sciences, Douglas College, New Westminster British Columbia, Canada
article info
abstract
Article history:
Background: Fungal and bacterial coinfections are common in surgical settings; however,
Received 10 August 2013
little is known about the effects of polymicrobial interactions on the cellular mechanisms
Received in revised form
involved in innate immune recognition and phagocytosis.
27 November 2013
Materials and methods: Zymosan particles, cell wall derivatives of the yeast Saccharomyces
Accepted 11 March 2014
cerevisiae, are used to model fungal interactions with host immune cells since they display
Available online 17 March 2014
carbohydrates, including beta-glucan, that are characteristic of fungal pathogens. Using in vitro cell culture, RAW 264.7 macrophages were challenged with zymosan, and phago-
Keywords:
cytosis determined via light microscopy. The effects of different concentrations of lipo-
Lipopolysaccharide (LPS)
polysaccharide (LPS) on zymosan phagocytosis were assessed. In addition, the transfer of
Macrophage Phagocytosis
supernatant from LPS-treated cells to naı¨ve cells, the effects of soluble carbohydrates laminarin, mannan, or galactomannan, and the impact of complement receptor 3 (CR3)
Zymosan
inhibition on phagocytosis were also determined.
Beta-glucans
Results: LPS enhanced phagocytosis of zymosan in a dose-dependent manner. Transfer of
CR3
supernatants from LPS-primed cells to naı¨ve cells had no effect on phagocytosis. Laminarin
Laminarin
inhibited zymosan phagocytosis in naı¨ve cells but not in LPS-primed cells. Neither mannan,
Opsonin
galactomannan, nor CR3 inhibition had a significant effect on ingestion of unopsonized
RAW 264.7
zymosan in naı¨ve or LPS-treated cells. Conclusions: Zymosan recognition by naı¨ve cells is inhibited by laminarin, but not mannan, galactomannan, or CR3 inhibition. LPS enhancement of phagocytosis is laminarin insensitive and not mediated by supernatant factors or zymosan engagement by the mannose or CR3 receptors. Our data suggest alternative mechanisms of zymosan recognition in the presence and absence of LPS. ª 2014 Elsevier Inc. All rights reserved.
* Corresponding author. Biology Department, Douglas College, P.O. Box 2503, New Westminster, B.C., Canada V3L 5B2. Tel.: þ1 604 527 5233; fax: þ1 604 527 5095. E-mail address: [email protected] (L. Sigola). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.03.024
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1.
Introduction
Fungal infections may cause minor illnesses involving superficial structures of the mucosae or integumentary system and more severe disease with infiltration of deep-seated tissues and/or systemic invasion. Severe fungal infections are more prevalent in patients immunocompromised by human immunodeficiency virus, patients with malignancies on antineoplastic chemotherapy, and transplant patients on immunosuppressants [1,2]. Infections with fungal pathogens, particularly Candida albicans and Candida glabrata, have increased in frequency in intensive care units [3] and commonly occur concomitantly with bacterial pathogens [4]. Furthermore, increased mortality rates due to postsurgical fungal sepsis correlate with the use of multiple antibiotics, prolonged use of antibiotics, and with concomitant bacterial sepsis [5], observations which suggest the importance of understanding immune responses to fungi in the context of simultaneous bacterial infection. Endotoxin is associated with enhanced proinflammatory mediator secretion and a cascade of reactions that often results in death [6]. In some situations, however, endotoxin exposure has been reported to augment antifungal resistance in the host. For example, when given before either inoculation with Aspergillus fumigatus or Cryptococcus neoformans, lipopolysaccharide (LPS) significantly reduced mortality and lowered fungal tissue burdens in mice [7,8]. Similar studies reported reduced C glabrata burdens in the tissues of mice exposed to either Escherichia coli or the bacterium’s LPS, compared with control mice [9]. Of additional note in this latter study, was the observation of enhanced C glabrata phagocytosis by macrophages exposed to LPS in vitro. C albicans phagocytosis is also increased in freshly isolated human peripheral blood monocytes after stimulation with LPS [10]. Significant progress has been made in understanding the host immune response to fungal pathogens. Macrophages contribute to fungal resistance through their role as professional phagocytes by seeking out, ingesting, and killing microbes; they also secrete chemokines and cytokines, release microbicides including nitric oxide and reactive oxygen intermediates, and activate adaptive T cell immunity after fungal component antigen processing and presentation [11e13]. Macrophage recognition of pathogens and their products is achieved by host expression of germline-encoded pattern recognition receptors (PRRs); these are soluble or membrane-bound receptors which recognize microorganisms at specific conserved targets called pathogen-associated molecular patterns (PAMPs) [14]. Beta-glucans are carbohydrates found in fungi, plants, and some bacteria [15]. In fungi, they serve as significant PAMPs for several species including C albicans [16], A fumigatus [17], Pneumocystis carinii [18], Coccidioides posadasii [19], S cerevisiae [20], and its cell wall derivative, zymosan. Zymosan is frequently used to model fungal recognition and phagocytosis by macrophages because it is composed of large amounts of beta-glucan, in addition to small quantities of mannose and chitin [21]. We have previously reported that laminarin, a soluble low molecular weight beta-glucan, has differential effects on zymosan phagocytosis in the absence and presence of LPS
305
[22]. Laminarin inhibited zymosan phagocytosis in the absence of LPS, consistent with beta-glucan serving as an important PAMP during macrophage recognition and phagocytosis of zymosan. The presence of LPS significantly enhanced zymosan phagocytosis, and laminarin failed to inhibit zymosan ingestion in cells primed with LPS, suggesting the presence of laminarin sensitive and insensitive mechanisms for macrophage recognition of zymosan in the absence and presence of LPS, respectively. In this study, we investigated the potential roles that any supernatant factors such as opsonins, the mannose receptor, or the complement receptor 3 (CR3) may play in contributing to the enhanced zymosan phagocytosis induced by LPS.
2.
Materials and methods
2.1.
Reagents
Zymosan prepared from S cerevisiae, laminarin from Laminaria digitata, mannan from S cerevisiae, galactomannan from the locust bean gum of Ceratonia siliqua, and LPS from E coli serotype 055:B5 were all obtained from Sigma-Aldrich (St. Louis, MO). To assess the role of the CR3 receptor in phagocytosis experiments, functional gradeepurified anti-mouse CD11b antibody M1/70 (Integrin aM or Mac-1a) and its isotype-matched rat antimouse IgG2b control antibody were obtained from eBioscience (San Diego, CA). For phagocytosis assays, macrophages were cultured in sterile tissue culture medium composed of Roswell Park Memorial Institute 1640 medium (Life Technologies, Burlington, ON), supplemented with 10% heat-inactivated fetal calf serum (Life Technologies), 100 U/mL penicillin (Life Technologies), 100 mg/mL streptomycin (Life Technologies), 25 mM N-2-hydroxyethylpiperazine-NV-2-ethanesulphonic acid (Life Technologies), and 10 mM L-glutamine (Life Technologies; R10). Dulbecco Modified Eagles Medium (DMEM) (Life Technologies) was used for the growth of RAW 264.7 macrophages and supplemented in the same way as the Roswell Park Memorial Institute to form D10. Sterile plasticware, noncharged, was obtained from Corning Inc (Fisher Scientific, Toronto, ON), and used for all experiments.
2.2.
Macrophage cell culture
RAW 264.7 macrophages from American Tissue Type Culture Collection were obtained from Cedarlane Laboratories, Burlington, ON. Cells were maintained in D10 and passaged every 3 d when >90% confluent. In preparation for phagocytosis assays, cells were harvested and centrifuged at room temperature for 7 min at 1100 rpm, resuspended in R10, counted, and viability assessed using trypan blue dye (Sigma-Aldrich, St. Louis, MO) exclusion. Viability in all experiments was >90%.
2.3.
Opsonization of zymosan
Serum opsonization of zymosan was achieved by incubating 1 108 zymosan particles per ml suspended in R10 with an equal volume of fetal calf serum at 37 C for 30 min with
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frequent shaking. The opsonized zymosan particles were then washed four times in R10 before use in phagocytosis assays.
2.4.
Macrophage phagocytosis assay
For phagocytosis assays, 1 105 RAW 264.7 macrophages in R10 medium were plated onto 12-mm glass coverslips (Fisher Scientific, Toronto, ON) placed in sterile 24-well tissue culture plates (Costar, Fisher Scientific, Toronto, ON), and macrophages allowed to adhere in a humidified 5% CO2 incubator for 30 min at 37 C. Zymosan particles were added to the cells at a zymosan to macrophage ratio of 10:1. Phagocytosis was allowed to proceed for 1 h at 37 C in a humidified 5% CO2 incubator. In experiments where the effects of either carbohydrates or anti-CD11b were assessed, cells were incubated in the presence of these reagents for 15 min before addition of zymosan. Phagocytosis was terminated by vigorously washing off noningested particles with warm R10 three times. Glass coverslips were air-dried for 15 min, stained using Wright stain (Sigma-Aldrich, St. Louis, MO) and then washed with water, then coverslips air-dried and mounted onto glass microscope slides using Permount (Fisher Scientific, Toronto, ON). Slides were examined by light microscopy at 400 or 600 magnification using a Nikon Eclipse 50i photomicroscope (Nikon Canada Inc, Richmond, BC). Phagocytosis was determined from photomicrographs at 400 or 600 magnification by counting at least 100 macrophages in at least four different fields of view for each experimental treatment. Phagocytosis was assessed as percentage of phagocytic cells (PP), mean number of particles per cell (MNP) and as phagocytic index (PI) determined as PI ¼ PP MNP. Photomicrographs of cells for illustrations were taken at 1000 magnification.
2.5.
Statistical analysis
Results are expressed as the mean standard error of the mean. Statistical analysis was performed using the VassarStat online program using either the unpaired two-tailed Student t-test or analysis of variance followed by the post hoc Tukey test, as appropriate. A P value of 0.05 was considered statistically significant. All experiments were repeated at least twice with similar results.
3.
Results
3.1. LPS enhances zymosan phagocytosis in a concentration-dependent manner To investigate the effect of LPS on zymosan phagocytosis, we compared ingestion of macrophages cultured with medium alone versus cells cultured with three different concentrations of LPS at 1, 10, and 100 ng/mL, respectively (Fig. 1). LPS increased all measures of phagocytosis, and its effects on MNP and PI were statistically significant (P ¼ 0.002 and P ¼ 0.0006, respectively). LPS treatment enhanced the efficiency of phagocytosis as reflected by an increase in MNP from 4.5 1.1 for cells cultured in medium alone to 17.5 3 for cells cultured with LPS 100 ng/mL (Fig. 1B). PI also
Fig. 1 e LPS enhances zymosan phagocytosis in a concentration-dependent manner. Cells (1 3 105) were cultured in either medium alone or LPS at 1, 10, and 100 ng/mL respectively, for 16 h before addition of zymosan particles at a macrophage-to-zymosan ratio of 1:10. Phagocytosis was allowed to proceed for 1 h at 37 C in a 5% CO2 incubator, after which phagocytosis was terminated, cells stained and phagocytosis determined by light microscopy and expressed as percentage phagocytosis (A), mean number of particles ingested per cell (B) and PI (C). The percentage of cells in either medium alone or LPS, ingesting 0e5, 6e10, 11e15, and >16 zymosan particles per cell was also determined (D). Photomicroscopy was performed under oil immersion at 31000 magnification (E) and shows representative cells cultured in either medium alone or specified concentrations of LPS for 16 h, and then cells presented with zymosan particles for 1 h. Scale bar on photomicrograph represents 10 mm. Results presented are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01 for specified samples versus medium alone.
increased significantly with LPS pretreatment. We also compared the effect of LPS on the percentage of cells ingesting various quantities of zymosan particles (Fig. 1D). Most (73%) of the naı¨ve cells cultured in medium alone
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Fig. 1 e (continued).
ingested 16 particles each. In contrast, treatment with LPS at 1, 10, and 100 ng/mL increased the percentage of cells with more than 16 zymosan particles each to 13%, 39%, and 53%, respectively. There was a progressive rise in the number of ingested particles within the cytoplasm of cells primed with increasing LPS concentrations, compared with naı¨ve cells cultured in medium alone (see photomicrographs in Fig. 1E). Together, these results suggest an increase in overall zymosan phagocytosis effected, in part, by augmentation of macrophage phagocytic avidity in association with rising LPS concentrations in cell cultures.
3.2. Supernatant transfer from LPS-stimulated cells to naı¨ve cells has little effect on zymosan phagocytosis We considered the possibility that supernatants from LPSprimed macrophages may contain cell-derived opsonins that contribute to the enhanced avidity for zymosan by cells pretreated with LPS for 16 h. To investigate this possibility, culture supernatants from cells primed with LPS for 16 h were removed and added to naı¨ve cells, and the effect of this transfer on phagocytosis compared with ingestion in cells cultured in either medium alone, with LPS for 16 h and supernatant retained, with LPS-stimulated cells from which the supernatant was washed off, and cells pretreated with LPS for only 20 min (see Fig. 2). Cells cultured for 16 h with LPS (0.5 ng/mL) showed significantly enhanced zymosan phagocytosis compared with cells cultured in medium alone (MNP for medium alone: 1.1 0.5 versus LPS 10.9 1.4). For cells cultured with LPS for 16 h from which the supernatants were washed off before zymosan addition, MNP was 8.6 0.1; although reduced when compared with cells primed for 16 h with LPS, this difference in MNP was not
statistically significant. For naı¨ve cells treated for 20 min with culture supernatants transferred from other cells primed with LPS for 16 h, MNP was 2.4 0.5, and thus similar to MNP observed in cells cultured in medium alone. Supernatant transfer from LPS 16 h cultures to naı¨ve cells also had little effect on PP or PI.
3.3. Laminarin, mannan, and galactomannan have little effects on LPS-induced augmentation of zymosan phagocytosis The surface of zymosan is rich in carbohydrates, particularly beta-glucan and mannose [21]. To assess the importance of carbohydrate recognition for zymosan phagocytosis, RAW 264.7 cells were exposed to zymosan in the presence or absence of soluble carbohydrates, specifically laminarin (a beta-glucan), mannan (a mannose receptor blocker), and galactomannan (a carbohydrate not present on zymosan; see Fig. 3A). Carbohydrates were added to naı¨ve cell cultures 15 min before addition of zymosan. Cells cultured in medium alone ingested zymosan with MNP of 3.7 1.2. The presence of mannan or galactomannan had little effect on phagocytosis (MNP 5.8 0.9 and 5.1 1.4, respectively). In contrast, laminarin suppressed phagocytosis of zymosan significantly, inhibiting MNP by 76% (MNP 0.9 0.5). Percentage phagocytosis and PI were similar in cells cultured in medium alone and with mannan or galactomannan, respectively; in contrast, laminarin significantly suppressed both percentage phagocytosis and PI (data not shown). We also examined the effects of these carbohydrates on the LPS-induced increase in phagocytosis (Fig. 3B). LPS significantly enhanced MNP (3.3 0.3 for medium alone versus 14.6 0.8 for LPS), and the presence of soluble carbohydrates in cell cultures did not ameliorate LPS modulation of zymosan ingestion.
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Fig. 2 e Supernatant transfer from LPS-primed cells to naı¨ve cells does not augment mean number of zymosan particles ingested. Cells (1 3 105) were cultured in either medium alone, with LPS 0.5 ng/mL for 16 h, with LPS for 16 h and supernatants (sups) washed off, with naı¨ve cells treated with supernatants from cells cultured with LPS for 16 h, or with LPS for 20 min before addition of zymosan particles at a macrophage-to-zymosan ratio of 1:10. Phagocytosis was allowed to proceed for 1 h at 37 C in a 5% CO2 incubator, after which phagocytosis was terminated, cells stained and viewed by light microscopy. Counts were performed and phagocytosis determined as percentage phagocytosis (A), mean number of particles ingested per cell (B) and PI (C). Results presented are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.0001, respectively, for specified samples versus medium alone.
Fig. 3 e Laminarin, mannan, and galactomannan have little effect on LPS-induced augmentation of zymosan phagocytosis. Cells (1 3 105) were cultured in either medium alone (A) or with LPS 1 ng/mL for 16 h (B), where indicated either laminarin, mannan, or galactomannan, respectively, each at 100 mg/mL, was added to cell cultures for 15 min before addition of zymosan particles at a macrophage-to-zymosan ratio of 1:10. Phagocytosis was allowed to proceed for 1 h at 37 C in a 5% CO2 incubator, after which phagocytosis was terminated, cells stained and phagocytosis determined by light microscopy and expressed as mean number of particles ingested per cell. Results presented are expressed as mean ± standard error of the mean. *P < 0.05 and **P < 0.01, respectively, for specified samples versus medium alone.
3.4. CR3 inhibition impairs phagocytosis of serumopsonized zymosan but has little impact on phagocytosis of unopsonized zymosan in the absence or presence of LPS The CR3 receptor has been reported to bind zymosan nonopsonically and mediate its recognition and subsequent phagocytosis [23]. Therefore, we examined the effect of specific CR3 blockade on zymosan ingestion in naı¨ve RAW cells and naı¨ve cells presented with serum-opsonized particles (see Fig. 4). Specific CR3 blockade with the anti-CD11b antibody
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M1/70 had little effect on ingestion of unopsonized zymosan by naı¨ve RAW cells, and this was similar for its isotypematched control antibody (MNP for medium alone: 2.2 0.2, M1/70: 2.2 0.2, and control antibody: 2.2 0.3). Similarly, CR3 inhibition had little effect on percentage phagocytosis and PI for unopsonized zymosan. Opsonization of zymosan with serum significantly enhanced phagocytosis by 132% (see Fig. 4B; MNP for unopsonized zymosan: 2.2 0.2 versus opsonized zymosan: 5.1 0.6). M1/70 significantly inhibited phagocytosis of opsonized zymosan by 35% (MNP 3.3 0.4), but the control antibody had little effect on MNP (opsonized zymosan: 5.1 0.6 versus control antibody plus opsonized zymosan: 6.5 1.1). CR3 inhibition also significantly suppressed PI for serum-opsonized zymosan. Finally, we compared the effects of CR3 inhibition, laminarin, and mannan on zymosan phagocytosis in naı¨ve cells and LPS-primed cells. As observed previously, naı¨ve cell ingestion of zymosan was significantly inhibited by laminarin (MNP for medium alone 4.3 0.8, MNP for laminarin 0.5 0.5), but mannan, CR3 inhibition, and the control antibody had little effect (3.1 0.7, 4 0.7, and 2.6 0.5, respectively; see Fig. 5A). We also studied the effect of CR3 receptor blockade on enhanced phagocytosis observed for unopsonized zymosan by macrophages primed for 16 h with LPS (see Fig. 5B). LPS significantly augmented zymosan ingestion (MNP for medium alone 4.3 0.8, MNP for LPS 9.9 0.5). The anti-CR3 and control antibodies had little effects respectively on the increased zymosan ingestion due to LPS (MNP for LPS plus M1/ 70: 12.4 2.8, MNP for LPS plus control antibody: 8.6 0.7), nor, as observed previously, was the LPS effect ameliorated by laminarin or mannan, which were used at higher concentrations of 400 mg/mL, respectively, in this experiment.
4.
Fig. 4 e CR3 inhibition impairs phagocytosis of serumopsonized zymosan but has little impact on phagocytosis of unopsonized zymosan. Cells (1 3 105) were cultured in either medium alone or with 20 mg/mL anti-CR3 antibody M1/70 or its isotype-matched control antibody (Ab) for 15 min at 4 C before addition of either unopsonized zymosan (dark bars) or serum-opsonized zymosan (light bars) at a macrophage-to-zymosan ratio of 1:10. Phagocytosis was allowed to proceed for 1 h at 37 C in a 5% CO2 incubator, after which phagocytosis was terminated, cells stained and phagocytosis determined by light microscopy and expressed as percentage phagocytosis (A), mean number of particles ingested per cell (B) and PI (C). Results presented are expressed as mean ± standard error mean. *P < 0.05 and ***P < 0.001, respectively.
Discussion
In this study we demonstrated that exposure of RAW 264.7 macrophages to LPS significantly enhanced the avidity and efficiency of zymosan phagocytosis in a concentrationdependent manner. Similar dose-dependent effects of LPS have been reported for human peripheral blood monocytes’ ingestion of C albicans [10] and increased phagocytic efficiency noted for RAW 264.7 cell uptake of E coli in macrophages exposed to lipid A [24]. We also confirmed that macrophage recognition of zymosan was laminarin sensitive in the absence of LPS but laminarin insensitive for LPS-primed cells [22]. Zymosan particles are composed primarily of betaglucan, and cell surface macrophage PRRs reported to bind to this carbohydrate include Dectin-1 [25], CR3 [26], SIGNR1 [27], scavenger receptors [28], and lactosylceramide [29]. The mannose receptor has also been implicated as a PRR for zymosan via recognition of mannan on these particles [30]. We investigated three possibilities that could account for increased phagocytosis in LPS-stimulated cells. First, the potential contribution of substances, such as opsonins, present in the supernatants of cells primed with LPS; second, the role of mannose receptors; and finally, we examined possible involvement of CR3.
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Fig. 5 e CR3 inhibition has little effect on phagocytosis of unopsonized zymosan in the presence of LPS. Cells were cultured in either medium alone (A) or pretreated with LPS at 1 ng/mL for 16 h (B). Fifteen minutes before the addition of zymosan particles at a macrophage-to-zymosan ratio of 1:10, cells were cultured with either laminarin (400 mg/mL), mannan (400 mg/mL), anti-CR3 antibody M1/70 (100 mg/mL) or its isotype-matched control antibody (100 mg/mL), and antibody pretreatments performed at 4 C. Phagocytosis was allowed to proceed for 1 h at 37 C in a 5% CO2 incubator, after which phagocytosis was terminated, cells stained and phagocytosis determined by light microscopy and expressed as mean number of particles ingested per cell. Results presented are expressed as mean ± standard error of the mean. **P < 0.01 for specified samples versus medium alone.
Zymosan binds soluble proteins, which may act as opsonins and these include Pentraxin 3 (PTX3) [31], C-reactive protein and serum amyloid protein [32]. Opsonins bind PAMPs on microorganisms, thus facilitating the recognition of numerous and varied microbes with the use of only a limited number of host receptors. Murine macrophages make very small quantities of C-reactive protein [33]; however they produce large amounts of PTX3 and serum amyloid protein in response to LPS stimulation [34,35]. Zymosan phagocytosis is enhanced in macrophages from PTX3 transgenic mice [31] whereas PTX3 binding to its targets is blocked by galactomannan [36]. In our experiments supernatant transfer from LPS-primed cells to naı¨ve cells had little effect on phagocytosis, and galactomannan had no effect on zymosan ingestion in neither naı¨ve nor LPS-stimulated cells.
Furthermore, the addition of exogenous PTX3 failed to increase zymosan uptake by cells (data not shown), suggesting little role for PTX3 during phagocytosis in our model. We noted a small decrease in MNP in cells primed overnight with LPS and where culture supernatants were removed, compared with cells where the supernatants were retained, but this reduction was not statistically significant. We speculate that LPS enhances the expression of particular cell surface receptors for zymosan and that opsonins play little role in LPSmediated augmentation of zymosan phagocytosis. Laminarin inhibited zymosan phagocytosis in cells cultured in medium alone, consistent with several reports on the significance of beta-glucans as PAMPs recognized by murine macrophages [37e39]. In the presence of LPS, however, we confirmed our previous finding that laminarin was ineffective in abrogating enhanced zymosan phagocytosis. Therefore, we examined the role of the mannose receptor in the LPSmediated increase in phagocytosis. Mannan binds mannose receptors [40] and SIGNR1 [41] and both PRRs contribute to zymosan recognition and phagocytosis. RAW cells lack SIGNR1 [42], and therefore this receptor likely does not explain the increase in zymosan phagocytosis due to LPS. In our experiments, mannan had little effect on ingestion of zymosan in cells cultured in the absence or presence of LPS, even when mannan concentrations as high as 1 mg/mL were used in cell cultures (data not shown). Li et al. [43] also showed that mannan had no effect on zymosan ingestion by murine resident peritoneal macrophages. CR3 also binds beta-glucans, and there is evidence in human neutrophils and macrophages [44], in addition to murine bone marrow derived macrophages [45], that CR3 is the primary PRR for this carbohydrate. Nonopsonic binding of zymosan to CR3 occurs via the lectin-binding site on CD11b [26]. Using the antiCD11b antibody M1/70, we abrogated the increase in phagocytosis of serum-opsonized zymosan over that of unopsonized particles, however M1/70 had no effect on either naı¨ve cell ingestion of unopsonized zymosan or LPS-induced augmentation of phagocytosis of unopsonized zymosan. This suggests little role for CR3 as a significant receptor for increased zymosan ingestion in cells pretreated with LPS. Our study suggests that RAW macrophages recognize betaglucan bearing particles via different mechanisms in the absence and presence of LPS. Similar to our observations with LPS, the toll-like receptor 9 ligand CpG enhanced phagocytosis of zymosan and C albicans, respectively [46], and the authors showed laminarin sensitive zymosan ingestion in naı¨ve cells, whereas cell primed with CpG increased zymosan intake but this was not ameliorated by laminarin. Much attention has focused on Dectin-1 as a zymosan receptor since Brown et al. [25] identified this C-type lectin (inhibited by laminarin) as the primary murine PRR for beta-glucans, although its expression is variable depending on cell type and maturation state [47]. In our study, we used RAW macrophages, which express little Dectin-1 [25]; furthermore, LPS has been reported to downregulate Dectin-1 expression in macrophages [48]. Based on these reports and our own observations, it is unlikely that Dectin-1 plays a major role in mediating the LPS effect on zymosan ingestion in RAW macrophages. However, in contrast to our findings and other studies, it is interesting to note that LPS-treated human monocytes have recently been
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reported to increase Dectin-1 transcription and cell surface expression with associated enhancement of C albicans phagocytosis [10]. Therefore, it seems possible that cell type and method of cell maturation may contribute to differences observed for LPS effects on Dectin-1 expression. Based on our data, it is unlikely that supernatants of LPSstimulated cells, the mannose receptor or CR3 recognition of zymosan, explain the enhancement of phagocytosis by LPS. Further investigation is required to examine what roles other receptors, including scavenger receptors and lactosylceramide, may play as alternative mechanisms of zymosan ingestion in cells previously exposed to LPS. Scavenger receptors are promiscuous transmembrane proteins expressed on a variety of cells, including macrophages, and they bind to a wide variety of targets such as apoptotic cells, microbes, and zymosan [28,49,50]. It is interesting to note that LPSmediated enhancement of C glabrata phagocytosis is associated with augmented expression of macrophage receptor with collagenous structure, a scavenger receptor recognized as a marker of macrophage activation [9]. Additional studies are required to determine what functional role macrophage receptor with collagenous structure, or other classes of scavenger receptors, may play in zymosan recognition and phagocytosis after exposure of cells to LPS. Lactosylceramide is a glycosphingolipid that contributes to the formation of plasma membrane lipid rafts and acts as a PRR that binds microbes, including C albicans [51]. It is predominantly expressed on neutrophil membranes and contributes to increased avidity of CR3 binding to nonopsonized zymosan [52]. Additional studies are required to examine any possible roles for this glycosphingolipid in LPS-mediated augmentation of zymosan ingestion by macrophages. The challenges of understanding innate immunity in the context of multiple interactions involving the host with commensal, symbiotic, and pathogenic microbes, in addition to intermicrobial cross talk, are intimately connected with recognizing the critical dynamic equilibrium that determines health and disease. There is mounting evidence for the influence of intestinal commensal bacteria in shaping innate immunity and for the importance of PRRs in the recognition of the microbiota for promotion of host-microbial symbioses [53,54]. These mutualistic interactions are not limited to bacteria and hosts, as there is also recent evidence for the importance of gut fungi in modulating bowel inflammation [55] and allergic reactions [56]. In the light of growing evidence of the importance of relationships between the immune system and microbiota in contributing to human health and disease, understanding the mechanisms of these interactions is critical for novel preventative and therapeutic approaches, especially when considering the growing prevalence of antimicrobial resistance and concomitant polymicrobial infections.
Acknowledgment This study was supported by the Douglas College Research and Scholarly Activity Fund. The work presented was carried out with the collaboration of all authors. A-.L.F. and L.S. designed the experiments, conducted the phagocytosis studies, and analyzed and
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interpreted the data. L.M. and J.V. maintained the cell line, stained slides, collected and analyzed the data, and contributed to discussions on data interpretation. All authors contributed to drafting the manuscript. A-.L.F. and L.S. critically reviewed and revised the manuscript.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
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