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Teleost soluble CSF-1R modulates cytokine profiles at an inflammatory site, and inhibits neutrophil chemotaxis, phagocytosis, and bacterial killing
Developmental and Comparative Immunology 49 (2015) 259–266
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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i
Teleost soluble CSF-1R modulates cytokine profiles at an inflammatory site, and inhibits neutrophil chemotaxis, phagocytosis, and bacterial killing Aja M. Rieger a, Jeffrey J. Havixbeck a, Miodrag Belosevic a,b, Daniel R. Barreda a,c,* a
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2P5, Canada School of Public Health, University of Alberta, Edmonton, Alberta T6G 2P5, Canada c Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada b
A R T I C L E
I N F O
Article history: Received 30 September 2014 Revised 28 November 2014 Accepted 1 December 2014 Available online 9 December 2014 Keywords: Phagocytosis Inflammation CSF-1 Soluble CSF-1R Neutrophil
A B S T R A C T
Soluble colony stimulating factor-1 receptor (sCSF-1R) is a novel bony fish protein that contributes to the regulation of macrophage proliferation. We recently showed that this soluble receptor is highly upregulated by teleost macrophages in the presence of apoptotic cells. Further, recombinant sCSF-1R inhibited leukocyte infiltration into a challenge site in vivo. Herein, we characterized the mechanisms underlying these changes as a platform to better understand the evolutionary origins of the CSF-1 immuneregulatory axis and inflammation control in teleosts. Using an in vivo model of self-resolving peritonitis, we show that sCSF-1R downregulates chemokine expression and inhibits neutrophil chemotaxis. Soluble CSF-1R also inhibited gene expression of several pro-inflammatory cytokines and promoted the expression of an anti-inflammatory mediator, IL-10. Finally, the phenotype of infiltrating neutrophils changed significantly in the presence of sCSF-1R. Both a reduced capacity for phagocytosis and pathogen killing were observed. Overall, our results implicate sCSF-1R as an important regulator of neutrophil responses in teleosts. It remains unclear whether this represents an inflammation regulatory factor that is unique to this animal group or one that may be evolutionarily conserved and continues to contribute to the regulation of antimicrobial processes at inflammatory sites in higher vertebrates. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Colony stimulating factor-1 (CSF-1; also referred to as the macrophage-colony stimulating factor) is the primary regulator of proliferation, differentiation, and survival of macrophages and their precursors (Barreda et al., 2004). It also controls key aspects of macrophage function including microbicidal activity (O’Mahony et al., 2008; Roilides et al., 1996), cytokine (Chitu and Stanley, 2006; Evans et al., 1995) and chemokine (Hashimoto et al., 1996; Sweet and Hume, 2003) production, chemotaxis (Boocock et al., 1989; Webb et al., 1996), and phagocytosis (Akagawa, 2002; Cheers et al., 1989). This is a testament to its broad importance to the regulation of host immunity. However, CSF-1 has also been linked to numerous pathological conditions, including but not limited to, allograft and xenograft rejection, cancer, autoimmune disorders, atherosclerosis, and obesity
Abbreviations: AC, apoptotic cell; PKM, primary kidney macrophage; sCSF-1R, soluble colony stimulating factor-1 receptor; ROS, reactive oxygen species; DHR, dihydrorhodamine; Q-PCR, quantitative PCR; SOCS3, suppressor of cytokine signaling 3. * Corresponding author. Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2P5, Canada. Tel.: +1 780 492 0375; fax: +1 780 492 4265. E-mail address: [email protected] (D.R. Barreda). http://dx.doi.org/10.1016/j.dci.2014.12.003 0145-305X/© 2014 Elsevier Ltd. All rights reserved.
(Chitu and Stanley, 2006; Kleemann et al., 2008; Lewis and Pollard, 2006; Menke et al., 2009; Rubin Kelley et al., 1994; Weisberg et al., 2003). As such, tight regulation of CSF-1 activity is critical to foster its beneficial immune-regulatory responses while minimizing the potential for deleterious outcomes. CSF-1 exerts its biological effects at nanomolar concentrations and several mechanisms have evolved to regulate its actions, including receptor-mediated endocytosis, metabolic processing and the inhibition of downstream signaling (Barreda et al., 2004; Rieger et al., 2014). CSF-1 activity is also regulated through intracellular modulation of gene expression of both CSF-1 and its cognate receptor CSF-1R (Barreda et al., 2004; Rieger et al., 2014). Most recently, we identified a unique mechanism for regulation of CSF-1 activity in teleost (bony) fish. These fish produce a novel soluble form of CSF-1 receptor (sCSF-1R), which decreases macrophage proliferation in a dose-dependent manner (Barreda and Belosevic, 2001; Barreda et al., 2005). The native protein was detected in goldfish serum (Barreda et al., 2005), providing early suggestions that this protein might play a role in systemic regulation of CSF-1 activity. With this in mind, we investigated the role of sCSF-1R in the regulation of teleost macrophage antimicrobial responses. In vitro, we found that soluble CSF-1R inhibited CSF-1-mediated reactive oxygen production, nitric oxide synthesis, chemotaxis, and phagocytosis in
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cultured macrophages (Grayfer et al., 2009). Further, in vivo experiments showed that this novel soluble protein inhibited zymosandriven leukocyte infiltration and ROS production in a dose-dependent manner (Rieger et al., 2013). In this study, we focused on the sCSF-1R-driven mechanisms that control neutrophil responses at a site of inflammation. We found that sCSF-1R inhibited neutrophil recruitment in vivo through downregulation of CXCL-8 expression, resulting in reduced neutrophil chemotaxis. Soluble CSF-1R also decreased expression of several pro-inflammatory cytokines that are well known to promote neutrophil activation and antimicrobial responses. Characterization of functional responses from inflammatory neutrophils showed a reduced capacity for phagocytosis and intracellular pathogen killing following intraperitoneal administration of sCSF-1R. Importantly, the contributions of sCSF-1R to the inhibition of neutrophil inflammatory responses showed marked similarities to those elicited by apoptotic cells (AC), implicating sCSF-1R as an important component of AC-driven inhibition of inflammation. However, broader contributions of AC to the global regulation of cytokine gene expression suggest that factors beyond sCSF-1R also contribute to the resolution phase of inflammation in this teleost fish. 2. Materials and methods 2.1. Animals Goldfish (Carassius auratus L.) 10–15 cm in length were purchased from Mt. Parnell Fisheries (Mercersburg, PA, USA) and imported to Canada through Aquatic Imports (Calgary, AB, CA). Fish were maintained in the Aquatic Facility of the Department of Biological Sciences, University of Alberta. The fish were held at 20 °C in a flow-through water system on a simulated natural photoperiod. All animals were maintained according to the guidelines of the Canadian Council on Animal Care. The University of Alberta Animal Care and Use Committee approved all protocols (ACUC-Biosciences; protocol number 149 and 706). 2.2. Goldfish primary kidney macrophage cultures As previously described (Neumann et al., 1998, 2000), primary kidney macrophages (PKM) were generated by seeding isolated leukocytes and culturing in 15 mL complete MGFL-15 medium (MGFL15 supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL gentamicin, 10% newborn calf serum (Gibco) and 5% carp serum), 5 mL of cell-conditioned medium from previous cultures, and incubated for 6–9 days at 20 °C. The cells were harvested from cultures during the proliferative phase of growth. 2.3. Isolation of goldfish leukocyte subsets To isolate goldfish immune cell subsets, isolated cells were layered onto 51% Percoll and centrifuged for 25 minutes at 400 × g and 4 °C. Mononuclear cells at the 51% Percoll/medium interface were collected and transferred into a new conical tube and washed twice with incomplete MGFL-15 medium via centrifugation for 10 minutes at 311 × g and 4 °C. All remaining liquid was decanted from the conical tube, leaving behind a pellet containing primarily red blood cells and neutrophils. Neutrophils were collected from this pellet as previously described (Katzenback and Belosevic, 2009; Rieger et al., 2012). Briefly, pelleted cells were treated with ACK Lysis buffer (Lonza) for 3 min to lyse contaminating red blood cells, washed twice with incomplete HBSS−/− via centrifugation for 10 minutes at 311 × g and 4 °C, and resuspended in complete HBSS−/− (HBSS supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 5% carp serum) in preparation for downstream assays.
2.4. Preparation of apoptotic cells Apoptotic cells were generated by incubating catfish 3B11 cells for 24 hours in the presence of 10 μg/mL cycloheximide as previously described (Rieger et al., 2012). Apoptotic 3B11 cells have been previously shown to generate responses similar to those induced by apoptotic goldfish kidney cells (Rieger et al., 2013); however, 3B11 mRNA is not recognized by goldfish Q-PCR primers, making them more amenable for these studies. Cells were subsequently washed twice in 1× PBS−/− (no calcium, no magnesium) in preparation for downstream assays. 2.5. Preparation of recombinant sCSF-1R Recombinant sCSF-1R was generated using a eukaryotic (insectbased) protein expression system because this allowed for production of a recombinant protein with proper folding and post-translational modifications, and minimized the potential for contamination with microbial products like LPS. The methodology used has been described previously (Barreda et al., 2005). In short, Drosophila KC embryonic cells were grown in ESF-921 medium (Expression Systems) at 27 °C. KC cells were transferred into Grace’s insect medium (Sigma), and transfected with 5 mg sCSF-1R expression plasmid using Cellfectin® Reagent (Invitrogen) according to the manufacturer’s instructions. Stable transfectants were selected with blasticidin at 75 μg mL−1. After two rounds of selection the stable cell lines were maintained in ESF-921 medium containing 10 μg mL−1 blasticidin for 5–6 days at 27 °C prior to passaging and collection of supernatants containing recombinant soluble CSF-1R. Recombinant sCSF-1R was engineered to contain a C-terminal 6xHis tag and V5 epitope to facilitate subsequent purification and detection protocols. KC/sCSF-1R cell line supernatants were dialyzed against NiNTA wash buffer and concentrated in preparation for purification. Recombinant protein was purified using Ni-NTA agarose columns (Qiagen) according to the manufacturer’s specifications. Purified proteins were eluted in Ni-NTA wash buffer, dialyzed extensively against 1× PBS −/−, and filter-sterilized in preparation for immunodetection and analysis of biological activity. Total protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Scientific) according to the manufacturer’s protocols. 2.6. In vivo effects of apoptotic cells, zymosan and sCSF-1R Goldfish were injected with 2.5 mg zymosan (Sigma) in 100 μL of 1× PBS−/− and either 5 × 106 apoptotic cells or 800 ng recombinant sCSF-1R for 24 hours (unless otherwise specified). Cells were then used in the following assays: 2.6.1. Phagocytosis assay Zymosan particles (Molecular Probes) were labeled overnight with 250 μg/mL FITC (Sigma) with continuous shaking at 4 °C in 1× PBS−/− (no magnesium, no calcium). Zymosan–FITC was added at a ratio of 5:1 (particle: cell) to 2 × 106 cells in incomplete MGFL-15 media (no serum) at 18 °C. Following phagocytosis, cells were fixed in 1% formaldehyde and analyzed by imaging flow cytometry. Analysis was performed using an ImageStreamx MkII (Amnis), as previously described (Rieger et al., 2010). Phagocytosis values represent the percentage of cells that contain internalized fluorescent zymosan particles. 2.6.2. Respiratory burst assay This assay was performed as previously described (Rieger et al., 2010, 2012, 2013). Briefly, following activation, cells were harvested and resuspended in 100 μL 1× PBS−/−. Dihydrorhodamine (DHR, Molecular Probes) was added to cells at a final concentration of 10 μM and incubated for 5 minutes to allow cells to take up
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the DHR. Phorbol 12-myristate 13-acetate (PMA; Sigma) was then added at a final concentration of 100 ng/mL. Cells were incubated for a further 30 minutes to allow oxidation of DHR. All samples were properly staggered with respect to timing to accommodate for the transient state of oxidized DHR fluorescence, measured by flow cytometry on a FACS Canto II (BD Biosciences). DHR was gated based on samples not stimulated with PMA. Responses above this basal level were considered positive. 2.6.3. Gene expression RNA was isolated from peritoneal lavage cells using a Qiagen RNeasy kit (Qiagen) and cDNA synthesis performed using SMARTScribe Reverse Transcriptase (Clontech) according to the manufacturer’s protocol. Quantitative PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR machine. RQ values were normalized against saline injected fish. Elongation factor 1 alpha (EF1α) was used as the endogenous control. Primer sequences can be found in Table 1. 2.6.4. Chemotaxis assay Chemotaxis assays were designed to examine the impact of soluble macrophage factors on inflammatory neutrophils. To examine macrophage-derived factors, PKM cultures were incubated with zymosan +/− increasing concentrations of recombinant sCSF-1R or apoptotic cells (5:1 ratio) for 24 hours. Supernatants were collected and filtered through a 0.22 μm filter. One hundred microliters of these supernatants were applied to the bottom chamber of a blind well leucite chemotaxis chamber (Nucleoprobe). Primary inflammatory neutrophils were derived from goldfish that had been injected with 2.5 mg zymosan (Sigma). Neutrophils were isolated from the goldfish peritoneal cavity after 24 hours by layering total cellular isolates over 51% Percoll, as described above in section 2.3. Neutrophils (1 × 106) were applied to the top chamber of a blind well leucite chemotaxis chamber and their migration into the bottom chamber observed. The top and bottom chambers were separated by a 5 μm pore polycarbonate membrane (NeuroProbe).
Table 1 Primer sequences for Q-PCR. PRIMER
SEQUENCE (5′-3′)
EF-1α forward EF-1α reverse CSF-1 forward CSF-1 reverse CXCL-8 forward CXCL-8 reverse IFNγ forward IFNγ reverse IFNγrel forward IFNγrel reverse IL-1β-1 forward IL-1β-1 reverse IL-1β-2 forward IL-1β-2 reverse IL-10 forward IL-10 reverse IL-12 p35 forward IL-12 p35 reverse IL-12 p40 forward IL-12 p40 reverse SOCS-3 forward SOCS-3 reverse TGFβ forward TGFβ reverse TNFα1 forward TNFα1 reverse TNFα2 forward TNFα2 reverse
CCG TTG AGA TGC ACC ATG AGT TTG ACA GAC ACG TTC TTC ACG TT ACA CAC ATA ACA GCC CAC AAA GCC AGC ACA GGA CAA GGA TGA AGC ACT CTG AGA GTC GAC GCA TTG GAA TGG TGT CTT TAC AGT GTG AGT TTG G GAA ACC CTA TGG GCG ATC AA GTA GAC ACG CTT CAG CTC AAA CA TGT CGG AGC CAG ACT TCC A GAC TCG ATT TTT TCT CGT ACG TTC T GCG CTG CTC AAC TTC ATC TTG GTG ACA CAT TAA GCG GCT TCA C GAT GCG CTG CTC AGC TTC T AGT GGG TGC TAC ATT AAC CAT ACG CAA GGA GCT CCG TTC TGC AT TCG AGT AAT GGT GCC AAG TCA TCA TGT TTT ACG TGC ATT CCT TTG G GGC GCC TGA AAA AAA TAC GA CTT CAG AAG CAG CTT TGT TGT TG CAG TTT TTG AGA GCT CACCGA TAT C CGA GTC GGG CAC CAA GAA AAG CTC TGG AGT CCG TCT GAA GTA CAC TAC GGC GGA GGA TTG CGC TTC GAT TCG CTT TCT CT CAT TCC TAC GGA TGG CAT TTA CTT CCT CAG GAA TGT CAG TCT TGC AT TCA TTC CTT ACG ACG GCA TTT CAG TCA CGT CAG CCT TGC AG
EF, elongation factor; SOCS, suppressor of cytokine signaling.
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Chemokinesis (ChK) controls utilized zymosan-stimulated supernatants in both the upper and lower chambers of the chemotaxis apparatus; these were used to assess neutrophil movement in the absence of a gradient. Cells were then incubated for 1 hour at 20 °C to allow for migration, after which cell suspensions were aspirated from the top chamber. Filters were removed, fixed in methanol, stained with Gill’s Solution 3 (Sigma) and then applied to a slide and fixed under a coverslip using Permount (Fisher Scientific). Chemotactic activity was assessed by counting cells found on the underside of filters in 20 random fields of view (100×, oil immersion). 2.6.5. Gentamicin protection assay Bacteria (Aeromonas veronii) was grown into log phase growth and added at a bacterial to cell ratio of 100:1. Cells were incubated with bacteria for 30 minutes, washed with incomplete media, resuspended in 100 μL incomplete media containing 200 μg/mL gentamicin and incubated for 30 minutes. After incubation, 2 mL of incomplete media was added to dilute the gentamicin. Cells were spun down and lysed with 90 μL MilliQ water with 0.1% Triton-X 100 for 30 seconds with vigorous flicking. Ten microliters of 10× PBS−/− was then added and lysate was plated onto square TSA plates following serial dilutions. Colony formation was counted after 48 hours. 2.7. Statistical analysis ImageStream data corresponding to the phagocytosis experiments were analyzed using IDEAS software (Amnis), as previously described (Rieger et al., 2010). Flow cytometry data corresponding to the respiratory burst experiments were acquired on a Canto II flow cytometer (BD Biosciences) and were analyzed using Diva or FCS Express v3 software. Statistics were performed as stated in figure legends using Prism 4 software (GraphPad Prism). 3. Results 3.1. Soluble CSF-1R inhibits chemokine expression and neutrophil chemotaxis We previously showed that recombinant sCSF-1R inhibited zymosan-induced leukocyte infiltration in vivo (Rieger et al., 2013). This effect was specific to this novel soluble cytokine receptor since a challenge with an irrelevant recombinant protein, the erythropoietin receptor, produced and purified under the same conditions did not result in any significant changes to leukocyte infiltration (Supplementary Fig. S1). The dose-dependent reduction in infiltration was associated with inflammatory neutrophils, in contrast to monocytes, macrophages and lymphocytes. Since neutrophils do not express CSF-1R and are not directly affected by CSF-1 (Rieger et al., 2013), we hypothesized that this inhibition in recruitment must rely on leukocyte cross talk. Given the central contribution of chemokines to leukocyte recruitment, we first examined the impact of sCSF1R on leukocyte expression of CXCL-8 and CCL-1, two important chemokines that promote neutrophil and monocyte/lymphocyte chemotaxis, respectively. Recombinant sCSF-1R significantly decreased gene expression of CXCL-8 among inflammatory leukocytes at all concentrations tested (Fig. 1). In comparison, a significant decrease in the expression of CCL-1 was limited to the highest concentration of sCSF-1R examined. Given that bioactive molecules secreted by macrophages are among the primary mediators of neutrophil recruitment to an inflammatory site, we tested whether sCSF-1R affected the capacity of macrophages to drive neutrophil chemotaxis. To assess this, we isolated infiltrating inflammatory neutrophils from challenged fish and measured chemotaxis toward supernatants from activated macrophages treated with increasing concentrations of sCSF-1R (Fig. 2).
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Fig. 1. Soluble CSF-1R alters chemokine expression, especially CXCL-8. Goldfish were injected i.p. with zymosan in the presence or absence of either apoptotic cells (AC) or recombinant sCSF-1R. After 24 hours, peritoneal cells were harvested by lavage and RNA was extracted. Chemokine levels were measured by Q-PCR. n = 6; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05 compared to zymosan determined by a oneway ANOVA (Tukey’s post-hoc). Graphs displayed with SEM.
The goal was to highlight the combinatorial effects of sCSF-1R on the production of macrophage factors that regulate neutrophil chemotaxis. Our results showed that neutrophil chemotaxis decreased in a dose-dependent manner with increasing concentrations of recombinant sCSF-1R, and reached similar levels to those achieved upon exposure of macrophages to apoptotic cells. 3.2. Soluble CSF-1R inhibits pro-inflammatory gene expression but not as broadly as apoptotic cells To further understand the mechanisms by which sCSF-1R inhibits acute inflammation in fish, we examined the expression of cytokine genes that are integral to the development and maintenance of inflammation and neutrophil function (Grayfer and Belosevic, 2009; Grayfer et al., 2008, 2009, 2010). Goldfish were injected with zymosan in the presence or absence of either sCSF-1R or apoptotic cells. Peritoneal cells were harvested by lavage after 24 hours and mRNA levels determined using Q-PCR. In addition to the chemokine findings described above, we found that sCSF-1R caused a significant decrease in the expression of TNF-α2, IL-1β1,
Fig. 2. Neutrophil chemotaxis is decreased by macrophage soluble factors produced in the presence of sCSF-1R. Goldfish primary macrophage cultures were incubated with zymosan in the presence/absence of increasing doses of recombinant sCSF-1R or apoptotic cells. After 24 hours, filtered supernatants were applied to the bottom of a blind-well chemotaxis chamber. Neutrophils isolated from zymosaninjected fish were isolated and applied to the top chamber. After 1 hour, membranes were removed and neutrophil chemotaxis was counted. ChK = chemokinesis control. n = 6; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05, ++p < 0.01 compared to zymosan determined by a one-way ANOVA (Tukey’s post-hoc). Graphs displayed with SEM.
IL-1β2, IFNγ, IFNγrel (IFNγ-related), IL12p35, and CSF-1 (Fig. 3). These decreases in pro-inflammatory cytokine expression were also observed upon addition of apoptotic cells, although with varying degrees of statistical significance. In mammals, the appearance of apoptotic cells at an inflammatory site is known to result in the production of anti-inflammatory soluble factors (Erwig and Henson, 2007, 2008; Huynh et al., 2002; Neufeld et al., 1999; Sanjabi et al., 2009; Tassiulas et al., 2007; Weigert et al., 2009). Thus, we examined whether sCSF-1R may also contribute to the resolution phase of inflammation through upregulation of anti-inflammatory cytokine gene expression. Among our panel of anti-inflammatory cytokines (IL-10, TGF-β, VEGF, and SOCS3), we only observed a significant increase in IL-10 expression following treatment with apoptotic cells (Fig. 4). In all cases examined, sCSF-1R did not lead to significant upregulation of antiinflammatory cytokine gene expression, suggesting that apoptotic cell-driven factors beyond sCSF-1R may also contribute to the resolution phase of inflammation in this teleost fish. To assess the potential for sCSF-1R dose-dependent immuneregulatory effects on cytokine gene expression, we challenged goldfish in vivo with zymosan in the presence or absence of apoptotic cells or increasing concentrations of sCSF-1R. We limited these studies to those cytokines that had shown the greatest significance in the experiments above (TNF-α2, CSF-1, IL-10, and SOCS3). We found that TNF-α2 mRNA levels decreased significantly at all concentrations examined for sCSF-1R (Fig. 5), suggesting that expression of TNF-α2 is highly responsive to the presence of sCSF-1R. In contrast, IL-10 showed a comparably narrow window for responsiveness to sCSF-1R, where mRNA levels only increased significantly upon in vivo administration of 200 ng sCSF-1R (Fig. 5). CSF-1 and SOCS3 showed similar dose responses, where cytokine gene expression decreased with increasing concentrations of sCSF1R (Fig. 5). In contrast, we did not identify significant changes in the expression of these cytokines among purified macrophage cultures suggesting that the cross talk between inflammatory leukocytes within the peritoneal cavity is an important component of cytokine gene expression in vivo (Supplementary Fig. S2).
3.3. Soluble CSF-1 receptor decreases anti-microbicidal neutrophil responses Upon entering the inflammatory site, mammalian neutrophils are rapidly activated by exposure to pro-inflammatory cytokines such as TNF-α and IL-1β, which enhance their capacity to defend the host against invading pathogens (Suzuki et al., 1999; Yuo et al., 1989). In the sections above, we showed that sCSF-1R reduced neutrophil recruitment in goldfish by modulating chemokine expression and neutrophil chemotaxis, and regulated pro- and antiinflammatory cytokine gene expression. We wondered if the presence of sCSF-1R may also impact neutrophil antimicrobial functions at the inflammatory site. To this end, goldfish were challenged with zymosan in vivo in the presence or absence of sCSF-1R or apoptotic cells, and neutrophils were isolated from the peritoneum and examined ex vivo (Supplementary Fig. S3). We found that neutrophil phagocytosis decreased significantly in fish injected with apoptotic cells or sCSF-1R (Fig. 6a). These responses were similar to those induced in macrophages, where both apoptotic cells and sCSF-1R also reduced macrophage phagocytosis (Supplementary Fig. S4a). Finally, because phagocytosis must be coupled to intracellular killing to result in effective pathogen clearance, inflammatory goldfish neutrophils were allowed to internalize live A. veronii. Consistent with the results presented above, incubation with apoptotic cells or sCSF-1R decreased the efficiency of bacterial killing (Fig. 6; 15 minute time point). Importantly, this decrease was more dramatic than that observed in macrophages, which exhibited a similar
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Fig. 3. Soluble CSF-1R inhibits gene expression for a range of pro-inflammatory cytokines. Goldfish were injected i.p. with zymosan in the presence or absence of either apoptotic cells (AC) or recombinant sCSF-1R. After 24 hours, peritoneal cells were harvested by lavage and RNA was extracted. Cytokine levels were measured by Q-PCR. Soluble CSF-1R causes a general downregulation of pro-inflammatory cytokines. n = 6; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05, ++p < 0.01 compared to zymosan; ‡p < 0.05 compared to zymosan plus apoptotic cells determined by a one-way ANOVA (Tukey’s post-hoc). Graphs displayed with SEM.
decrease but at later time points (Supplementary Fig. S4b; 60 minute time point). 4. Discussion Inflammation is an important physiological process for the clearance and control of pathogens. However, a loss or untimely control of inflammation can have grave consequences for the host since it can contribute to the development of chronic inflammatory diseases, autoimmunity, and chronic infection (Duffield, 2003; Poon et al., 2010; Soehnlein and Lindbom, 2010). Macrophages and their central regulator, CSF-1, have been tightly linked to both the beneficial and deleterious effects of inflammation (Chitu and Stanley, 2006). As such, the CSF-1 regulatory system represents an important node for the control of inflammation. Yet, almost 40 years after the original discovery of CSF-1, we continue to identify new strategies for control of CSF-1 activity and become increasingly aware of the potential positive and negative repercussions if it is to be used as a therapeutic target. In this manuscript we add to the evolutionary insights into the regulation of CSF-1 activity using a teleost fish model. Soluble receptors have been shown to be important and common regulators of many cytokines within the immune system, including IL-1, IL-6, IFNγ, TNF-α, GM-CSF, G-CSF and others (Fernandez-Botran,
1991; Heaney and Golde, 1998). We previously described a novel soluble receptor, sCSF-1R, that to date has only been found in teleost fish. In primary macrophage cultures, sCSF-1R was found in highest levels during periods of significant inhibition of macrophage proliferation and differentiation. In the senescent phase of goldfish in vitro macrophage development, increases in macrophage production of sCSF-1R paralleled the induction of apoptotic events (Barreda et al., 2005). Further, recombinant sCSF-1R inhibited macrophage proliferation pointing to a potential role in hematopoiesis control (Barreda et al., 2005). Aside from its direct effect on macrophages, our results in this study show that this novel soluble protein also contributes to the control of teleost fish inflammation by modulating neutrophil activity. We found that CXCL-8, a chemokine known to be involved in neutrophil recruiting, was greatly decreased at all concentrations of sCSF-1R. Similarly to what has been described in mammals (Harada et al., 1994), CXCL-8 may be the primary chemokine involved in neutrophil recruitment in goldfish. While CXCL-8 was greatly affected, we also found significant decreases in CCL-1, a recruiter of monocytes and lymphocytes. The mRNA expression data presented in this study matched closely with our previously published results, where sCSF-1R treatment inhibited monocyte, macrophage, and lymphocyte infiltration (Rieger et al., 2013). The changes in chemokine expression described above paralleled a significant inhibition in the expression pro-inflammatory
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by sCSF-1R and apoptotic cells significantly reduced the capacity of neutrophils to clear phagocytosed bacteria. This likely reflects the importance of a timely inflammation control response both for the effective clearance of attacking pathogens and the induction of tissue repair/homeostatic programs. Overall, the proper control of inflammation is of outmost physiological importance for the long-term survival of a host. In this manuscript we provide important mechanistic insights into the control of inflammation by a teleost soluble CSF-1 receptor. This soluble receptor affects every aspect of an inflammatory response examined – the ability to recruit cells into an inflammatory site, production of inflammatory cytokines and chemokines, and effector mechanisms at the inflammatory site. This speaks to the importance of CSF-1 to inflammation control and suggests that its contributions are broadly conserved across vertebrates. Further, it is interesting that sCSF-1R can recapitulate most of the antiinflammatory functions induced by apoptotic cells in goldfish, placing it as a primary component of the anti-inflammatory effects that are elicited by apoptotic cells in this species. It will be interesting then to determine whether sCSF-1R will be equally effective at recapitulating other downstream resolution and the tissue repair mechanisms that are initiated by apoptosis at the inflammatory site. Acknowledgements Fig. 4. Both apoptotic cells and sCSF-1R have a limited impact on anti-inflammatory cytokine expression. Goldfish were injected i.p. with zymosan in the presence or absence of either apoptotic cells (AC) or recombinant sCSF-1R. After 24 hours, peritoneal cells were harvested by lavage and RNA was extracted. Cytokine levels were measured by Q-PCR. n = 6; *p < 0.05 compared to PBS; ‡p < 0.05 compared to zymosan plus apoptotic cells determined by a one-way ANOVA (Tukey’s post-hoc). Graphs displayed with SEM.
cytokines including IL-1β1 and IL-1β 2, IFNγ and IFNγrel, TNF-α2, IL-12p35 and CSF-1. All of these have been previously implicated in the regulation of goldfish macrophage pro-inflammatory responses, especially phagocytosis and the production of reactive intermediates (Grayfer and Belosevic, 2009; Grayfer et al., 2008, 2009, 2010). Notably, for TNFα2 and CSF-1 we identified a differential decrease in mRNA expression at the lowest sCSF-1R concentrations tested. It will be interesting to examine the impact of low doses of sCSF-1R across a panel of pro-inflammatory cytokines to assess whether this is a general phenomenon highlighting a differential level of sensitivity to sCSF-1R. When we studied canonical anti-inflammatory responses, we found further differences between responses to sCSF-1R and apoptotic cells. The most dramatic effect was seen on IL-10 expression. Unlike the pro-inflammatory cytokines, we found that an increase in IL-10 expression was limited to lower concentrations of sCSF-1R. Interestingly, our work did not show an increase in TGF-β following apoptotic cell exposure, even though this represents the primary mediator of anti-inflammatory responses following phagocytosis of apoptotic cells in mammals (Fadok et al., 1998). It is possible that sCSF-1R may exert differential responses at other concentrations for TGF-β and other antiinflammatory cytokines, or that additional or different triggers may be required to induce production following exposure to apoptotic cells in teleost fish. Activation of neutrophil responses is highly dependent on the soluble factors that they are exposed to upon entering an inflammatory site. Thus, we examined if changes to complex in vivo milieu induced by apoptotic cell or sCSF-1R could affect neutrophilspecific inflammatory responses. We found that both the presence of sCSF-1R and apoptotic cells significantly reduced neutrophil phagocytosis. Finally, the changes to the inflammatory milieu induced
We thank Jeffrey D. Konowalchuk and John Sony Robbins for their technical assistance. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) grants to DRB (RGPIN-2013-355303) and MB (RGPIN-2014-96395). AMR was supported by NSERC Vanier Doctoral Scholarship and a University of Alberta Dissertation Fellowship. JJH was supported by an NSERC
Fig. 5. Soluble CSF-1R has dose-dependent effects on important pro-, antiinflammatory and chemotactic factors. Goldfish were injected i.p. with zymosan in the presence or absence of either apoptotic cells (AC) or recombinant sCSF-1R. After 24 hours, peritoneal cells were harvested by lavage and RNA was extracted. Cytokine levels were measured by Q-PCR. n = 6; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05, ++p < 0.01 compared to zymosan determined by a one-way ANOVA (Tukey’s posthoc). Graphs displayed with SEM.
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Fig. 6. Neutrophil killing responses are affected by soluble products produced by macrophages treated with sCSF-1R. Goldfish were injected i.p. with zymosan in the presence or absence of either apoptotic cells (AC) or recombinant sCSF-1R. After 24 hours, peritoneal cells were harvested by lavage and neutrophils were isolated. (a) Cells were then incubated with zymosan-FITC particles (5:1 ratio, particle:cell) for 2 hours to measure phagocytosis. n = 5; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05, ++p < 0.01 compared to zymosan determined by one-way ANOVA (Tukey’s posthoc) (b) Neutrophil killing potential was assessed with a gentamicin protection assay. Neutrophils were incubated in the soluble products from PKMs activated with the noted stimuli for 2 hours. Aeromonas veronii was then added (100:1) for 30 minutes. Cells were then treated with gentamicin for 30 minutes and lysed at the indicated time points. n = 3; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05, ++p < 0.01 compared to zymosan determined by a two-way ANOVA (Bonferroni post-hoc). Graphs displayed with SEM.
PGS-M, a University of Alberta teaching assistantship, and an NSERC Vanier Doctoral Scholarship.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.dci.2014.12.003.
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Contents lists available at ScienceDirect
Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i
Teleost soluble CSF-1R modulates cytokine profiles at an inflammatory site, and inhibits neutrophil chemotaxis, phagocytosis, and bacterial killing Aja M. Rieger a, Jeffrey J. Havixbeck a, Miodrag Belosevic a,b, Daniel R. Barreda a,c,* a
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2P5, Canada School of Public Health, University of Alberta, Edmonton, Alberta T6G 2P5, Canada c Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada b
A R T I C L E
I N F O
Article history: Received 30 September 2014 Revised 28 November 2014 Accepted 1 December 2014 Available online 9 December 2014 Keywords: Phagocytosis Inflammation CSF-1 Soluble CSF-1R Neutrophil
A B S T R A C T
Soluble colony stimulating factor-1 receptor (sCSF-1R) is a novel bony fish protein that contributes to the regulation of macrophage proliferation. We recently showed that this soluble receptor is highly upregulated by teleost macrophages in the presence of apoptotic cells. Further, recombinant sCSF-1R inhibited leukocyte infiltration into a challenge site in vivo. Herein, we characterized the mechanisms underlying these changes as a platform to better understand the evolutionary origins of the CSF-1 immuneregulatory axis and inflammation control in teleosts. Using an in vivo model of self-resolving peritonitis, we show that sCSF-1R downregulates chemokine expression and inhibits neutrophil chemotaxis. Soluble CSF-1R also inhibited gene expression of several pro-inflammatory cytokines and promoted the expression of an anti-inflammatory mediator, IL-10. Finally, the phenotype of infiltrating neutrophils changed significantly in the presence of sCSF-1R. Both a reduced capacity for phagocytosis and pathogen killing were observed. Overall, our results implicate sCSF-1R as an important regulator of neutrophil responses in teleosts. It remains unclear whether this represents an inflammation regulatory factor that is unique to this animal group or one that may be evolutionarily conserved and continues to contribute to the regulation of antimicrobial processes at inflammatory sites in higher vertebrates. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Colony stimulating factor-1 (CSF-1; also referred to as the macrophage-colony stimulating factor) is the primary regulator of proliferation, differentiation, and survival of macrophages and their precursors (Barreda et al., 2004). It also controls key aspects of macrophage function including microbicidal activity (O’Mahony et al., 2008; Roilides et al., 1996), cytokine (Chitu and Stanley, 2006; Evans et al., 1995) and chemokine (Hashimoto et al., 1996; Sweet and Hume, 2003) production, chemotaxis (Boocock et al., 1989; Webb et al., 1996), and phagocytosis (Akagawa, 2002; Cheers et al., 1989). This is a testament to its broad importance to the regulation of host immunity. However, CSF-1 has also been linked to numerous pathological conditions, including but not limited to, allograft and xenograft rejection, cancer, autoimmune disorders, atherosclerosis, and obesity
Abbreviations: AC, apoptotic cell; PKM, primary kidney macrophage; sCSF-1R, soluble colony stimulating factor-1 receptor; ROS, reactive oxygen species; DHR, dihydrorhodamine; Q-PCR, quantitative PCR; SOCS3, suppressor of cytokine signaling 3. * Corresponding author. Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2P5, Canada. Tel.: +1 780 492 0375; fax: +1 780 492 4265. E-mail address: [email protected] (D.R. Barreda). http://dx.doi.org/10.1016/j.dci.2014.12.003 0145-305X/© 2014 Elsevier Ltd. All rights reserved.
(Chitu and Stanley, 2006; Kleemann et al., 2008; Lewis and Pollard, 2006; Menke et al., 2009; Rubin Kelley et al., 1994; Weisberg et al., 2003). As such, tight regulation of CSF-1 activity is critical to foster its beneficial immune-regulatory responses while minimizing the potential for deleterious outcomes. CSF-1 exerts its biological effects at nanomolar concentrations and several mechanisms have evolved to regulate its actions, including receptor-mediated endocytosis, metabolic processing and the inhibition of downstream signaling (Barreda et al., 2004; Rieger et al., 2014). CSF-1 activity is also regulated through intracellular modulation of gene expression of both CSF-1 and its cognate receptor CSF-1R (Barreda et al., 2004; Rieger et al., 2014). Most recently, we identified a unique mechanism for regulation of CSF-1 activity in teleost (bony) fish. These fish produce a novel soluble form of CSF-1 receptor (sCSF-1R), which decreases macrophage proliferation in a dose-dependent manner (Barreda and Belosevic, 2001; Barreda et al., 2005). The native protein was detected in goldfish serum (Barreda et al., 2005), providing early suggestions that this protein might play a role in systemic regulation of CSF-1 activity. With this in mind, we investigated the role of sCSF-1R in the regulation of teleost macrophage antimicrobial responses. In vitro, we found that soluble CSF-1R inhibited CSF-1-mediated reactive oxygen production, nitric oxide synthesis, chemotaxis, and phagocytosis in
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cultured macrophages (Grayfer et al., 2009). Further, in vivo experiments showed that this novel soluble protein inhibited zymosandriven leukocyte infiltration and ROS production in a dose-dependent manner (Rieger et al., 2013). In this study, we focused on the sCSF-1R-driven mechanisms that control neutrophil responses at a site of inflammation. We found that sCSF-1R inhibited neutrophil recruitment in vivo through downregulation of CXCL-8 expression, resulting in reduced neutrophil chemotaxis. Soluble CSF-1R also decreased expression of several pro-inflammatory cytokines that are well known to promote neutrophil activation and antimicrobial responses. Characterization of functional responses from inflammatory neutrophils showed a reduced capacity for phagocytosis and intracellular pathogen killing following intraperitoneal administration of sCSF-1R. Importantly, the contributions of sCSF-1R to the inhibition of neutrophil inflammatory responses showed marked similarities to those elicited by apoptotic cells (AC), implicating sCSF-1R as an important component of AC-driven inhibition of inflammation. However, broader contributions of AC to the global regulation of cytokine gene expression suggest that factors beyond sCSF-1R also contribute to the resolution phase of inflammation in this teleost fish. 2. Materials and methods 2.1. Animals Goldfish (Carassius auratus L.) 10–15 cm in length were purchased from Mt. Parnell Fisheries (Mercersburg, PA, USA) and imported to Canada through Aquatic Imports (Calgary, AB, CA). Fish were maintained in the Aquatic Facility of the Department of Biological Sciences, University of Alberta. The fish were held at 20 °C in a flow-through water system on a simulated natural photoperiod. All animals were maintained according to the guidelines of the Canadian Council on Animal Care. The University of Alberta Animal Care and Use Committee approved all protocols (ACUC-Biosciences; protocol number 149 and 706). 2.2. Goldfish primary kidney macrophage cultures As previously described (Neumann et al., 1998, 2000), primary kidney macrophages (PKM) were generated by seeding isolated leukocytes and culturing in 15 mL complete MGFL-15 medium (MGFL15 supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL gentamicin, 10% newborn calf serum (Gibco) and 5% carp serum), 5 mL of cell-conditioned medium from previous cultures, and incubated for 6–9 days at 20 °C. The cells were harvested from cultures during the proliferative phase of growth. 2.3. Isolation of goldfish leukocyte subsets To isolate goldfish immune cell subsets, isolated cells were layered onto 51% Percoll and centrifuged for 25 minutes at 400 × g and 4 °C. Mononuclear cells at the 51% Percoll/medium interface were collected and transferred into a new conical tube and washed twice with incomplete MGFL-15 medium via centrifugation for 10 minutes at 311 × g and 4 °C. All remaining liquid was decanted from the conical tube, leaving behind a pellet containing primarily red blood cells and neutrophils. Neutrophils were collected from this pellet as previously described (Katzenback and Belosevic, 2009; Rieger et al., 2012). Briefly, pelleted cells were treated with ACK Lysis buffer (Lonza) for 3 min to lyse contaminating red blood cells, washed twice with incomplete HBSS−/− via centrifugation for 10 minutes at 311 × g and 4 °C, and resuspended in complete HBSS−/− (HBSS supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 5% carp serum) in preparation for downstream assays.
2.4. Preparation of apoptotic cells Apoptotic cells were generated by incubating catfish 3B11 cells for 24 hours in the presence of 10 μg/mL cycloheximide as previously described (Rieger et al., 2012). Apoptotic 3B11 cells have been previously shown to generate responses similar to those induced by apoptotic goldfish kidney cells (Rieger et al., 2013); however, 3B11 mRNA is not recognized by goldfish Q-PCR primers, making them more amenable for these studies. Cells were subsequently washed twice in 1× PBS−/− (no calcium, no magnesium) in preparation for downstream assays. 2.5. Preparation of recombinant sCSF-1R Recombinant sCSF-1R was generated using a eukaryotic (insectbased) protein expression system because this allowed for production of a recombinant protein with proper folding and post-translational modifications, and minimized the potential for contamination with microbial products like LPS. The methodology used has been described previously (Barreda et al., 2005). In short, Drosophila KC embryonic cells were grown in ESF-921 medium (Expression Systems) at 27 °C. KC cells were transferred into Grace’s insect medium (Sigma), and transfected with 5 mg sCSF-1R expression plasmid using Cellfectin® Reagent (Invitrogen) according to the manufacturer’s instructions. Stable transfectants were selected with blasticidin at 75 μg mL−1. After two rounds of selection the stable cell lines were maintained in ESF-921 medium containing 10 μg mL−1 blasticidin for 5–6 days at 27 °C prior to passaging and collection of supernatants containing recombinant soluble CSF-1R. Recombinant sCSF-1R was engineered to contain a C-terminal 6xHis tag and V5 epitope to facilitate subsequent purification and detection protocols. KC/sCSF-1R cell line supernatants were dialyzed against NiNTA wash buffer and concentrated in preparation for purification. Recombinant protein was purified using Ni-NTA agarose columns (Qiagen) according to the manufacturer’s specifications. Purified proteins were eluted in Ni-NTA wash buffer, dialyzed extensively against 1× PBS −/−, and filter-sterilized in preparation for immunodetection and analysis of biological activity. Total protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Scientific) according to the manufacturer’s protocols. 2.6. In vivo effects of apoptotic cells, zymosan and sCSF-1R Goldfish were injected with 2.5 mg zymosan (Sigma) in 100 μL of 1× PBS−/− and either 5 × 106 apoptotic cells or 800 ng recombinant sCSF-1R for 24 hours (unless otherwise specified). Cells were then used in the following assays: 2.6.1. Phagocytosis assay Zymosan particles (Molecular Probes) were labeled overnight with 250 μg/mL FITC (Sigma) with continuous shaking at 4 °C in 1× PBS−/− (no magnesium, no calcium). Zymosan–FITC was added at a ratio of 5:1 (particle: cell) to 2 × 106 cells in incomplete MGFL-15 media (no serum) at 18 °C. Following phagocytosis, cells were fixed in 1% formaldehyde and analyzed by imaging flow cytometry. Analysis was performed using an ImageStreamx MkII (Amnis), as previously described (Rieger et al., 2010). Phagocytosis values represent the percentage of cells that contain internalized fluorescent zymosan particles. 2.6.2. Respiratory burst assay This assay was performed as previously described (Rieger et al., 2010, 2012, 2013). Briefly, following activation, cells were harvested and resuspended in 100 μL 1× PBS−/−. Dihydrorhodamine (DHR, Molecular Probes) was added to cells at a final concentration of 10 μM and incubated for 5 minutes to allow cells to take up
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the DHR. Phorbol 12-myristate 13-acetate (PMA; Sigma) was then added at a final concentration of 100 ng/mL. Cells were incubated for a further 30 minutes to allow oxidation of DHR. All samples were properly staggered with respect to timing to accommodate for the transient state of oxidized DHR fluorescence, measured by flow cytometry on a FACS Canto II (BD Biosciences). DHR was gated based on samples not stimulated with PMA. Responses above this basal level were considered positive. 2.6.3. Gene expression RNA was isolated from peritoneal lavage cells using a Qiagen RNeasy kit (Qiagen) and cDNA synthesis performed using SMARTScribe Reverse Transcriptase (Clontech) according to the manufacturer’s protocol. Quantitative PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR machine. RQ values were normalized against saline injected fish. Elongation factor 1 alpha (EF1α) was used as the endogenous control. Primer sequences can be found in Table 1. 2.6.4. Chemotaxis assay Chemotaxis assays were designed to examine the impact of soluble macrophage factors on inflammatory neutrophils. To examine macrophage-derived factors, PKM cultures were incubated with zymosan +/− increasing concentrations of recombinant sCSF-1R or apoptotic cells (5:1 ratio) for 24 hours. Supernatants were collected and filtered through a 0.22 μm filter. One hundred microliters of these supernatants were applied to the bottom chamber of a blind well leucite chemotaxis chamber (Nucleoprobe). Primary inflammatory neutrophils were derived from goldfish that had been injected with 2.5 mg zymosan (Sigma). Neutrophils were isolated from the goldfish peritoneal cavity after 24 hours by layering total cellular isolates over 51% Percoll, as described above in section 2.3. Neutrophils (1 × 106) were applied to the top chamber of a blind well leucite chemotaxis chamber and their migration into the bottom chamber observed. The top and bottom chambers were separated by a 5 μm pore polycarbonate membrane (NeuroProbe).
Table 1 Primer sequences for Q-PCR. PRIMER
SEQUENCE (5′-3′)
EF-1α forward EF-1α reverse CSF-1 forward CSF-1 reverse CXCL-8 forward CXCL-8 reverse IFNγ forward IFNγ reverse IFNγrel forward IFNγrel reverse IL-1β-1 forward IL-1β-1 reverse IL-1β-2 forward IL-1β-2 reverse IL-10 forward IL-10 reverse IL-12 p35 forward IL-12 p35 reverse IL-12 p40 forward IL-12 p40 reverse SOCS-3 forward SOCS-3 reverse TGFβ forward TGFβ reverse TNFα1 forward TNFα1 reverse TNFα2 forward TNFα2 reverse
CCG TTG AGA TGC ACC ATG AGT TTG ACA GAC ACG TTC TTC ACG TT ACA CAC ATA ACA GCC CAC AAA GCC AGC ACA GGA CAA GGA TGA AGC ACT CTG AGA GTC GAC GCA TTG GAA TGG TGT CTT TAC AGT GTG AGT TTG G GAA ACC CTA TGG GCG ATC AA GTA GAC ACG CTT CAG CTC AAA CA TGT CGG AGC CAG ACT TCC A GAC TCG ATT TTT TCT CGT ACG TTC T GCG CTG CTC AAC TTC ATC TTG GTG ACA CAT TAA GCG GCT TCA C GAT GCG CTG CTC AGC TTC T AGT GGG TGC TAC ATT AAC CAT ACG CAA GGA GCT CCG TTC TGC AT TCG AGT AAT GGT GCC AAG TCA TCA TGT TTT ACG TGC ATT CCT TTG G GGC GCC TGA AAA AAA TAC GA CTT CAG AAG CAG CTT TGT TGT TG CAG TTT TTG AGA GCT CACCGA TAT C CGA GTC GGG CAC CAA GAA AAG CTC TGG AGT CCG TCT GAA GTA CAC TAC GGC GGA GGA TTG CGC TTC GAT TCG CTT TCT CT CAT TCC TAC GGA TGG CAT TTA CTT CCT CAG GAA TGT CAG TCT TGC AT TCA TTC CTT ACG ACG GCA TTT CAG TCA CGT CAG CCT TGC AG
EF, elongation factor; SOCS, suppressor of cytokine signaling.
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Chemokinesis (ChK) controls utilized zymosan-stimulated supernatants in both the upper and lower chambers of the chemotaxis apparatus; these were used to assess neutrophil movement in the absence of a gradient. Cells were then incubated for 1 hour at 20 °C to allow for migration, after which cell suspensions were aspirated from the top chamber. Filters were removed, fixed in methanol, stained with Gill’s Solution 3 (Sigma) and then applied to a slide and fixed under a coverslip using Permount (Fisher Scientific). Chemotactic activity was assessed by counting cells found on the underside of filters in 20 random fields of view (100×, oil immersion). 2.6.5. Gentamicin protection assay Bacteria (Aeromonas veronii) was grown into log phase growth and added at a bacterial to cell ratio of 100:1. Cells were incubated with bacteria for 30 minutes, washed with incomplete media, resuspended in 100 μL incomplete media containing 200 μg/mL gentamicin and incubated for 30 minutes. After incubation, 2 mL of incomplete media was added to dilute the gentamicin. Cells were spun down and lysed with 90 μL MilliQ water with 0.1% Triton-X 100 for 30 seconds with vigorous flicking. Ten microliters of 10× PBS−/− was then added and lysate was plated onto square TSA plates following serial dilutions. Colony formation was counted after 48 hours. 2.7. Statistical analysis ImageStream data corresponding to the phagocytosis experiments were analyzed using IDEAS software (Amnis), as previously described (Rieger et al., 2010). Flow cytometry data corresponding to the respiratory burst experiments were acquired on a Canto II flow cytometer (BD Biosciences) and were analyzed using Diva or FCS Express v3 software. Statistics were performed as stated in figure legends using Prism 4 software (GraphPad Prism). 3. Results 3.1. Soluble CSF-1R inhibits chemokine expression and neutrophil chemotaxis We previously showed that recombinant sCSF-1R inhibited zymosan-induced leukocyte infiltration in vivo (Rieger et al., 2013). This effect was specific to this novel soluble cytokine receptor since a challenge with an irrelevant recombinant protein, the erythropoietin receptor, produced and purified under the same conditions did not result in any significant changes to leukocyte infiltration (Supplementary Fig. S1). The dose-dependent reduction in infiltration was associated with inflammatory neutrophils, in contrast to monocytes, macrophages and lymphocytes. Since neutrophils do not express CSF-1R and are not directly affected by CSF-1 (Rieger et al., 2013), we hypothesized that this inhibition in recruitment must rely on leukocyte cross talk. Given the central contribution of chemokines to leukocyte recruitment, we first examined the impact of sCSF1R on leukocyte expression of CXCL-8 and CCL-1, two important chemokines that promote neutrophil and monocyte/lymphocyte chemotaxis, respectively. Recombinant sCSF-1R significantly decreased gene expression of CXCL-8 among inflammatory leukocytes at all concentrations tested (Fig. 1). In comparison, a significant decrease in the expression of CCL-1 was limited to the highest concentration of sCSF-1R examined. Given that bioactive molecules secreted by macrophages are among the primary mediators of neutrophil recruitment to an inflammatory site, we tested whether sCSF-1R affected the capacity of macrophages to drive neutrophil chemotaxis. To assess this, we isolated infiltrating inflammatory neutrophils from challenged fish and measured chemotaxis toward supernatants from activated macrophages treated with increasing concentrations of sCSF-1R (Fig. 2).
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Fig. 1. Soluble CSF-1R alters chemokine expression, especially CXCL-8. Goldfish were injected i.p. with zymosan in the presence or absence of either apoptotic cells (AC) or recombinant sCSF-1R. After 24 hours, peritoneal cells were harvested by lavage and RNA was extracted. Chemokine levels were measured by Q-PCR. n = 6; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05 compared to zymosan determined by a oneway ANOVA (Tukey’s post-hoc). Graphs displayed with SEM.
The goal was to highlight the combinatorial effects of sCSF-1R on the production of macrophage factors that regulate neutrophil chemotaxis. Our results showed that neutrophil chemotaxis decreased in a dose-dependent manner with increasing concentrations of recombinant sCSF-1R, and reached similar levels to those achieved upon exposure of macrophages to apoptotic cells. 3.2. Soluble CSF-1R inhibits pro-inflammatory gene expression but not as broadly as apoptotic cells To further understand the mechanisms by which sCSF-1R inhibits acute inflammation in fish, we examined the expression of cytokine genes that are integral to the development and maintenance of inflammation and neutrophil function (Grayfer and Belosevic, 2009; Grayfer et al., 2008, 2009, 2010). Goldfish were injected with zymosan in the presence or absence of either sCSF-1R or apoptotic cells. Peritoneal cells were harvested by lavage after 24 hours and mRNA levels determined using Q-PCR. In addition to the chemokine findings described above, we found that sCSF-1R caused a significant decrease in the expression of TNF-α2, IL-1β1,
Fig. 2. Neutrophil chemotaxis is decreased by macrophage soluble factors produced in the presence of sCSF-1R. Goldfish primary macrophage cultures were incubated with zymosan in the presence/absence of increasing doses of recombinant sCSF-1R or apoptotic cells. After 24 hours, filtered supernatants were applied to the bottom of a blind-well chemotaxis chamber. Neutrophils isolated from zymosaninjected fish were isolated and applied to the top chamber. After 1 hour, membranes were removed and neutrophil chemotaxis was counted. ChK = chemokinesis control. n = 6; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05, ++p < 0.01 compared to zymosan determined by a one-way ANOVA (Tukey’s post-hoc). Graphs displayed with SEM.
IL-1β2, IFNγ, IFNγrel (IFNγ-related), IL12p35, and CSF-1 (Fig. 3). These decreases in pro-inflammatory cytokine expression were also observed upon addition of apoptotic cells, although with varying degrees of statistical significance. In mammals, the appearance of apoptotic cells at an inflammatory site is known to result in the production of anti-inflammatory soluble factors (Erwig and Henson, 2007, 2008; Huynh et al., 2002; Neufeld et al., 1999; Sanjabi et al., 2009; Tassiulas et al., 2007; Weigert et al., 2009). Thus, we examined whether sCSF-1R may also contribute to the resolution phase of inflammation through upregulation of anti-inflammatory cytokine gene expression. Among our panel of anti-inflammatory cytokines (IL-10, TGF-β, VEGF, and SOCS3), we only observed a significant increase in IL-10 expression following treatment with apoptotic cells (Fig. 4). In all cases examined, sCSF-1R did not lead to significant upregulation of antiinflammatory cytokine gene expression, suggesting that apoptotic cell-driven factors beyond sCSF-1R may also contribute to the resolution phase of inflammation in this teleost fish. To assess the potential for sCSF-1R dose-dependent immuneregulatory effects on cytokine gene expression, we challenged goldfish in vivo with zymosan in the presence or absence of apoptotic cells or increasing concentrations of sCSF-1R. We limited these studies to those cytokines that had shown the greatest significance in the experiments above (TNF-α2, CSF-1, IL-10, and SOCS3). We found that TNF-α2 mRNA levels decreased significantly at all concentrations examined for sCSF-1R (Fig. 5), suggesting that expression of TNF-α2 is highly responsive to the presence of sCSF-1R. In contrast, IL-10 showed a comparably narrow window for responsiveness to sCSF-1R, where mRNA levels only increased significantly upon in vivo administration of 200 ng sCSF-1R (Fig. 5). CSF-1 and SOCS3 showed similar dose responses, where cytokine gene expression decreased with increasing concentrations of sCSF1R (Fig. 5). In contrast, we did not identify significant changes in the expression of these cytokines among purified macrophage cultures suggesting that the cross talk between inflammatory leukocytes within the peritoneal cavity is an important component of cytokine gene expression in vivo (Supplementary Fig. S2).
3.3. Soluble CSF-1 receptor decreases anti-microbicidal neutrophil responses Upon entering the inflammatory site, mammalian neutrophils are rapidly activated by exposure to pro-inflammatory cytokines such as TNF-α and IL-1β, which enhance their capacity to defend the host against invading pathogens (Suzuki et al., 1999; Yuo et al., 1989). In the sections above, we showed that sCSF-1R reduced neutrophil recruitment in goldfish by modulating chemokine expression and neutrophil chemotaxis, and regulated pro- and antiinflammatory cytokine gene expression. We wondered if the presence of sCSF-1R may also impact neutrophil antimicrobial functions at the inflammatory site. To this end, goldfish were challenged with zymosan in vivo in the presence or absence of sCSF-1R or apoptotic cells, and neutrophils were isolated from the peritoneum and examined ex vivo (Supplementary Fig. S3). We found that neutrophil phagocytosis decreased significantly in fish injected with apoptotic cells or sCSF-1R (Fig. 6a). These responses were similar to those induced in macrophages, where both apoptotic cells and sCSF-1R also reduced macrophage phagocytosis (Supplementary Fig. S4a). Finally, because phagocytosis must be coupled to intracellular killing to result in effective pathogen clearance, inflammatory goldfish neutrophils were allowed to internalize live A. veronii. Consistent with the results presented above, incubation with apoptotic cells or sCSF-1R decreased the efficiency of bacterial killing (Fig. 6; 15 minute time point). Importantly, this decrease was more dramatic than that observed in macrophages, which exhibited a similar
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Fig. 3. Soluble CSF-1R inhibits gene expression for a range of pro-inflammatory cytokines. Goldfish were injected i.p. with zymosan in the presence or absence of either apoptotic cells (AC) or recombinant sCSF-1R. After 24 hours, peritoneal cells were harvested by lavage and RNA was extracted. Cytokine levels were measured by Q-PCR. Soluble CSF-1R causes a general downregulation of pro-inflammatory cytokines. n = 6; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05, ++p < 0.01 compared to zymosan; ‡p < 0.05 compared to zymosan plus apoptotic cells determined by a one-way ANOVA (Tukey’s post-hoc). Graphs displayed with SEM.
decrease but at later time points (Supplementary Fig. S4b; 60 minute time point). 4. Discussion Inflammation is an important physiological process for the clearance and control of pathogens. However, a loss or untimely control of inflammation can have grave consequences for the host since it can contribute to the development of chronic inflammatory diseases, autoimmunity, and chronic infection (Duffield, 2003; Poon et al., 2010; Soehnlein and Lindbom, 2010). Macrophages and their central regulator, CSF-1, have been tightly linked to both the beneficial and deleterious effects of inflammation (Chitu and Stanley, 2006). As such, the CSF-1 regulatory system represents an important node for the control of inflammation. Yet, almost 40 years after the original discovery of CSF-1, we continue to identify new strategies for control of CSF-1 activity and become increasingly aware of the potential positive and negative repercussions if it is to be used as a therapeutic target. In this manuscript we add to the evolutionary insights into the regulation of CSF-1 activity using a teleost fish model. Soluble receptors have been shown to be important and common regulators of many cytokines within the immune system, including IL-1, IL-6, IFNγ, TNF-α, GM-CSF, G-CSF and others (Fernandez-Botran,
1991; Heaney and Golde, 1998). We previously described a novel soluble receptor, sCSF-1R, that to date has only been found in teleost fish. In primary macrophage cultures, sCSF-1R was found in highest levels during periods of significant inhibition of macrophage proliferation and differentiation. In the senescent phase of goldfish in vitro macrophage development, increases in macrophage production of sCSF-1R paralleled the induction of apoptotic events (Barreda et al., 2005). Further, recombinant sCSF-1R inhibited macrophage proliferation pointing to a potential role in hematopoiesis control (Barreda et al., 2005). Aside from its direct effect on macrophages, our results in this study show that this novel soluble protein also contributes to the control of teleost fish inflammation by modulating neutrophil activity. We found that CXCL-8, a chemokine known to be involved in neutrophil recruiting, was greatly decreased at all concentrations of sCSF-1R. Similarly to what has been described in mammals (Harada et al., 1994), CXCL-8 may be the primary chemokine involved in neutrophil recruitment in goldfish. While CXCL-8 was greatly affected, we also found significant decreases in CCL-1, a recruiter of monocytes and lymphocytes. The mRNA expression data presented in this study matched closely with our previously published results, where sCSF-1R treatment inhibited monocyte, macrophage, and lymphocyte infiltration (Rieger et al., 2013). The changes in chemokine expression described above paralleled a significant inhibition in the expression pro-inflammatory
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by sCSF-1R and apoptotic cells significantly reduced the capacity of neutrophils to clear phagocytosed bacteria. This likely reflects the importance of a timely inflammation control response both for the effective clearance of attacking pathogens and the induction of tissue repair/homeostatic programs. Overall, the proper control of inflammation is of outmost physiological importance for the long-term survival of a host. In this manuscript we provide important mechanistic insights into the control of inflammation by a teleost soluble CSF-1 receptor. This soluble receptor affects every aspect of an inflammatory response examined – the ability to recruit cells into an inflammatory site, production of inflammatory cytokines and chemokines, and effector mechanisms at the inflammatory site. This speaks to the importance of CSF-1 to inflammation control and suggests that its contributions are broadly conserved across vertebrates. Further, it is interesting that sCSF-1R can recapitulate most of the antiinflammatory functions induced by apoptotic cells in goldfish, placing it as a primary component of the anti-inflammatory effects that are elicited by apoptotic cells in this species. It will be interesting then to determine whether sCSF-1R will be equally effective at recapitulating other downstream resolution and the tissue repair mechanisms that are initiated by apoptosis at the inflammatory site. Acknowledgements Fig. 4. Both apoptotic cells and sCSF-1R have a limited impact on anti-inflammatory cytokine expression. Goldfish were injected i.p. with zymosan in the presence or absence of either apoptotic cells (AC) or recombinant sCSF-1R. After 24 hours, peritoneal cells were harvested by lavage and RNA was extracted. Cytokine levels were measured by Q-PCR. n = 6; *p < 0.05 compared to PBS; ‡p < 0.05 compared to zymosan plus apoptotic cells determined by a one-way ANOVA (Tukey’s post-hoc). Graphs displayed with SEM.
cytokines including IL-1β1 and IL-1β 2, IFNγ and IFNγrel, TNF-α2, IL-12p35 and CSF-1. All of these have been previously implicated in the regulation of goldfish macrophage pro-inflammatory responses, especially phagocytosis and the production of reactive intermediates (Grayfer and Belosevic, 2009; Grayfer et al., 2008, 2009, 2010). Notably, for TNFα2 and CSF-1 we identified a differential decrease in mRNA expression at the lowest sCSF-1R concentrations tested. It will be interesting to examine the impact of low doses of sCSF-1R across a panel of pro-inflammatory cytokines to assess whether this is a general phenomenon highlighting a differential level of sensitivity to sCSF-1R. When we studied canonical anti-inflammatory responses, we found further differences between responses to sCSF-1R and apoptotic cells. The most dramatic effect was seen on IL-10 expression. Unlike the pro-inflammatory cytokines, we found that an increase in IL-10 expression was limited to lower concentrations of sCSF-1R. Interestingly, our work did not show an increase in TGF-β following apoptotic cell exposure, even though this represents the primary mediator of anti-inflammatory responses following phagocytosis of apoptotic cells in mammals (Fadok et al., 1998). It is possible that sCSF-1R may exert differential responses at other concentrations for TGF-β and other antiinflammatory cytokines, or that additional or different triggers may be required to induce production following exposure to apoptotic cells in teleost fish. Activation of neutrophil responses is highly dependent on the soluble factors that they are exposed to upon entering an inflammatory site. Thus, we examined if changes to complex in vivo milieu induced by apoptotic cell or sCSF-1R could affect neutrophilspecific inflammatory responses. We found that both the presence of sCSF-1R and apoptotic cells significantly reduced neutrophil phagocytosis. Finally, the changes to the inflammatory milieu induced
We thank Jeffrey D. Konowalchuk and John Sony Robbins for their technical assistance. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) grants to DRB (RGPIN-2013-355303) and MB (RGPIN-2014-96395). AMR was supported by NSERC Vanier Doctoral Scholarship and a University of Alberta Dissertation Fellowship. JJH was supported by an NSERC
Fig. 5. Soluble CSF-1R has dose-dependent effects on important pro-, antiinflammatory and chemotactic factors. Goldfish were injected i.p. with zymosan in the presence or absence of either apoptotic cells (AC) or recombinant sCSF-1R. After 24 hours, peritoneal cells were harvested by lavage and RNA was extracted. Cytokine levels were measured by Q-PCR. n = 6; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05, ++p < 0.01 compared to zymosan determined by a one-way ANOVA (Tukey’s posthoc). Graphs displayed with SEM.
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Fig. 6. Neutrophil killing responses are affected by soluble products produced by macrophages treated with sCSF-1R. Goldfish were injected i.p. with zymosan in the presence or absence of either apoptotic cells (AC) or recombinant sCSF-1R. After 24 hours, peritoneal cells were harvested by lavage and neutrophils were isolated. (a) Cells were then incubated with zymosan-FITC particles (5:1 ratio, particle:cell) for 2 hours to measure phagocytosis. n = 5; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05, ++p < 0.01 compared to zymosan determined by one-way ANOVA (Tukey’s posthoc) (b) Neutrophil killing potential was assessed with a gentamicin protection assay. Neutrophils were incubated in the soluble products from PKMs activated with the noted stimuli for 2 hours. Aeromonas veronii was then added (100:1) for 30 minutes. Cells were then treated with gentamicin for 30 minutes and lysed at the indicated time points. n = 3; *p < 0.05, **p < 0.01 compared to PBS; +p < 0.05, ++p < 0.01 compared to zymosan determined by a two-way ANOVA (Bonferroni post-hoc). Graphs displayed with SEM.
PGS-M, a University of Alberta teaching assistantship, and an NSERC Vanier Doctoral Scholarship.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.dci.2014.12.003.
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