Abstract
In the current study, we investigated the effects of carp Il10 on phagocytes and lymphocytes. Carp Il10 shares several prototypical inhibitory activities on phagocytes with mammalian IL-10, including deactivation of neutrophils and macrophages, as shown by inhibition of oxygen and nitrogen radical production, as well as reduced expression of proinflammatory genes and mhc genes involved in Ag presentation. Similar to mammalian IL-10, carp Il10 acts through a signaling pathway involving phosphorylation of Stat3, ultimately leading to the early upregulation of socs3 expression. To our knowledge, this is the first study of the effects of Il10 on lymphocytes in fish. Although Il10 did not affect survival and proliferation of T cells from naive animals, it greatly promoted survival and proliferation of T cells in cultures from immunized animals, but only when used in combination with the immunizing Ag. Preliminary gene expression analysis suggests that, under these circumstances, carp Il10 stimulates a subset of CD8+ memory T cells while downregulating CD4+ memory Th1 and Th2 responses. In addition to the regulatory effect on T cells, carp Il10 stimulates proliferation, differentiation, and Ab secretion by IgM+ B cells. Overall, carp Il10 shares several prototypical activities with mammalian IL-10, including downregulation of the inflammatory response of phagocytes, stimulation of proliferation of subsets of memory T lymphocytes, and proliferation, differentiation, and Ab secretion by IgM+ B lymphocytes. To our knowledge, this is the first comprehensive analysis of biological activities of fish Il10 on both phagocytes and lymphocytes showing functional conservation of several properties of Il10.
Introduction
Interleukin-10, first named “cytokine synthesis inhibitory factor” because of its potent inhibitory effect on IL-2 and IFN-γ synthesis in Th1 cell clones, was first identified in mouse Th2 cell clones (1). It is recognized that IL-10 is produced by almost all leukocyte subtypes, but CD4+ T cells and monocytes/macrophages are considered the most important sources of this cytokine (2). The production of IL-10 is highly regulated, and its actual cellular source depends on the kind of stimulus, affected tissue, and phase of the immune response (3). IL-10 is considered a pleiotropic regulatory cytokine and one of the most important anti-inflammatory cytokines whose function is to limit and, ultimately, terminate the immune response, preventing the damaging effects of inflammation.
IL-10 acts on different cell populations from both the innate and adaptive branches of the immune system. Monocytes/macrophages are recognized as its main target to directly inhibit the synthesis of proinflammatory cytokines, reactive radical species, surface expression of molecules involved in Ag presentation, and phagocytosis (4). By affecting Ag presentation and cytokine production in APCs, IL-10 indirectly limits the activation and effector functions of Th cells. In a manner very similar to the one observed in macrophages, IL-10 also exerts a direct, potent, and rapid shutdown of neutrophils (5). The downregulation of proinflammatory activities in innate immune cells induced by IL-10 is the first step toward an anti-inflammatory or regulatory state of immunity.
IL-10 also exerts anti-inflammatory activities on the adaptive branch of the immune system, with direct inhibitory effects on the proliferation of CD4+ T cells (6), as well as on cytokine synthesis by Th1 cells (IL-2 and IFN-γ) and by Th2 cells (IL-4 and IL-5) (7, 8), but it does not seem to have a direct effect on cytokine synthesis by Th17 cells (IL-17) (9). IL-10 also indirectly affects the development or resolution of adaptive immune responses via its general inhibition of cytokine synthesis [e.g., by affecting the development of Th1 and Th17 immunity (10, 11)]. The downregulation of T cell proliferation can be considered the second step toward developing an anti-inflammatory or regulatory state of immunity.
Although IL-10 may induce a general and aspecific shutdown of the innate immune system and induce a specific shutdown of T cell–mediated responses, stimulatory roles for IL-10 also were described: IL-10 prevents apoptosis, increases proliferation and MHC class II expression in B cells, and plays a stimulatory role in Ig class switching (12, 13). Other stimulatory effects of IL-10 include the increase in cytotoxic activity of NK cells (14) and the induction of proliferation of certain subsets of CD8+ T cells (15). Altogether, IL-10 is a cytokine with a key role in the termination of inflammatory responses and successful restoration of homeostasis characterized by the development of long-lived memory cells to face future threats.
IL-10 acts as a homodimer and signals via the IL-10R complex. Two IL-10 homodimers bind four molecules of IL-10R1 to finally recruit IL-10R2 (16). Both receptors belong to the class II cytokine receptor family; although IL-10R1 is specific for IL-10, IL-10R2 also can act as a coreceptor for several other cytokines (5). After binding to its receptor complex, IL-10 activates a JAK/STAT-signaling pathway that, in most cases, leads to the activation of JAK1 (associated with IL-10R1) and TYK2 (associated with IL-10R2), as well as to subsequent phosphorylation of the transcription factor STAT3. Finally, phosphorylated STAT3 dimerizes and translocates to the nucleus where it starts the transcription of several genes, among which SOCS3 is considered ultimately responsible for the inhibition of cytokine synthesis (17).
The above-described activation routes of IL-10 and its inhibitory effects are based on knowledge acquired from studies in mammals, but not much is known about the function of IL-10 in lower vertebrates. Sequences coding for il10 homologs in teleosts were described for several fish species (18–26), including rainbow trout (Oncorhynchus mykiss), goldfish (Carassius auratus), zebrafish (Danio rerio), and common carp (Cyprinus carpio). In most fish species, il10 is primarily expressed in head kidney (bone marrow equivalent), spleen, and gills and is upregulated upon administration of LPS (20, 23, 25, 27). Recombinant goldfish Il10 was shown to reduce radical production in monocytes and inhibit transcription of specific proinflammatory cytokines (23). Grass carp Il10, induced by TGF-β1, increased cell viability of PBLs (24). Regretfully, in the latter study, the effects on B or T lymphocytes in the PBL population were not characterized. In general, although structural analysis of the protein sequence of fish Il10 indicated that sites and motifs essential for its bioactivity and tertiary structure are well conserved, not much is known about its function in fish.
In the current study, recombinant carp Il10 was used to assess its activity on cells typical of the innate and adaptive immune systems. Indeed, carp Il10 exerted the prototypical anti-inflammatory activities on macrophages and neutrophils that are seen in mammals: it downregulated oxygen and nitrogen radical production and cytokine synthesis by inhibiting proinflammatory gene expression. Further, we describe the differential effects of Il10 on the activation of B and T cells in fish for the first time to our knowledge. Carp Il10 strongly stimulated proliferation of Ag-specific memory T cells (most likely CD8+) and IgM+ B cells. Furthermore, Il10 promoted total IgM secretion, while also promoting maturation and differentiation of IgM+ B cells: in peripheral blood–derived cultures, Il10 promoted differentiation and proliferation of mature B cells (mBs), whereas in head kidney leukocyte (HKL)–derived cultures, Il10 stimulated the differentiation of Ag-specific plasma cells (PCs), leading to increased secretion of Ag-specific IgM in culture supernatants from immunized fish. This study describes novel and unique data about the bioactivity of fish Il10 on naive and memory B and T cells and characterizes the effect of this cytokine on B cell development. This report contributes to a complete overview of the bioactivity of this cytokine on phagocytes and lymphocytes in fish and its functional conservation throughout evolution.
Materials and Methods
Production of recombinant carp Il10
The il10 sequence (JX524550) coding for the mature carp Il10 protein without signal peptide was amplified from carp head kidney cDNA using specific primers (Table I) and cloned between the BamHI (5′ end) and HindIII (3′ end) sites of the pQE-30UA expression vector (QIAGEN). Ligation products were cloned in M15-competent Escherichia coli, plated onto Lysogeny broth-ampicillin-kanamycin plates, and incubated overnight at 37°C. Positive clones were identified by colony PCR using vector-specific primers, and the products were sequenced to verify correct orientation and frame.
Selected positive clones were used to inoculate 200 ml Lysogeny broth-ampicillin-kanamycin overnight culture. Then, the culture was used to inoculate 4 l Terrific broth-ampicillin-kanamycin medium, which was incubated until an OD600 of 0.6–0.9 was reached. Protein expression was induced with 1 mM IPTG. After 4 h of incubation at 37°C, bacteria were pelleted and lysed by adding 0.1 mg/ml lysozyme in 0.05 M Tris, 0.5 M NaCl for 30 min at room temperature (RT). The reaction was stopped by adding 0.02 M imidazole, 0.01 M DTT, 0.005 M EDTA, and 1% Triton X-100 (v/v), followed by four cycles of freeze-thawing. MgCl2 was added to a final concentration of 0.05 M, and a benzonase treatment (2 U/ml) was performed for 30 min at RT. Inclusion bodies were pelleted and solubilized in 0.1 M Tris-HCl, 6 M guanidine hydrochloride, and 20 mM imidazole (pH 8.5). To stimulate refolding of the protein, samples were incubated at RT for 30 min with 20 mM DTT and for 20 min with 30 mM oxidized glutathione disulfide and were diluted rapidly 10 times with 50 mM Tris-HCl (pH 10.7) and 500 mM NaCl. l-cysteine (6 mM) was added, and the samples were incubated overnight at 4°C with gentle stirring. Insoluble precipitate was removed by centrifugation (10,000 × g, 20 min, 4°C), and refolded proteins were purified using a Ni-NTA agarose column (QIAGEN). Briefly, the column was washed with 5× the column volume of buffer Ia (0.05 M Tris, 0.5 M NaCl, 0.025 M imidazole, 1% [v/v] Triton X-100), 30× the column volume of cold buffer Ib (0.05 M Tris, 0.5 M NaCl, 0.025 M imidazole, 1% [v/v] Triton X-114), 5× the column volume of buffer IIb (0.05 M Tris, 0.5 M NaCl, 0.025 M imidazole, 40% [v/v] propanol), and 7× the column volume of wash buffer (500 mM NaCl, 2 mM KCl, 20 mM Na2HPO4, 2 mM KH2PO4, 0.025 M imidazole). The proteins were eluted with elution buffer (500 mM NaCl, 2 mM KCl, 20 mM Na2HPO4, 2 mM KH2PO4, 0.25 M imidazole). Eluted proteins were dialyzed against PBS, filtered sterilized, mixed with 20% (v/v) glycerol, and stored at −80°C until further use. All of the steps were carried out using endotoxin-free materials. The purity of the protein was assessed by SDS-PAGE and Western blotting, and the concentration was assessed using a Nanodrop 1000 spectrometer (Thermo Scientific).
Titration of recombinant carp Il10
The recombinant protein was titrated using a reporter assay. Briefly, the EPC cell line from the cyprinid fish fathead minnow (28) was transfected with 3.5 μg pNiFty-Luc, a plasmid encoding the luciferase reporter gene under the control of the NF-κB–inducible ELAM-1 composite promoter (InvivoGen). Stably transfected cells (EPC–NF-κB–Luc) were selected using 250 μg/ml Zeocin (Life Technologies). EPC–NF-κB–Luc (50,000 cells/well) in Advanced DMEM/F-12 supplemented with 5% FBS (Life Technologies) was seeded in a 96-well plate, incubated overnight at 27°C, and stimulated with 50 μg/ml LPS (from E. coli 055:B5, Sigma-Aldrich) and different concentrations of recombinant protein (1.5–50 μg/ml) in a final volume of 100 μl complete medium. After 6 h at 27°C, cells were lysed using Bright glow (Promega), and the suspension was transferred to white 96-well plates with an opaque bottom (Corning), after which the luminescence was measured using a FilterMax F5 Multi-Mode Microplate Reader (Molecular Devices). Samples stimulated with LPS only yielded the maximum luminescence and were set to 100%; control samples incubated with medium only were considered 0% activation. One unit of Il10 was defined as the amount of recombinant protein needed to inhibit 50% of the maximum LPS-induced luciferase expression.
Fish and immunization
European common carp (C. carpio carpio L.) were bred and raised in the central fish facility Carus at Wageningen University at 23°C in recirculating UV-treated tap water and fed pelleted dry food (Sniff, Soest, Germany) daily. R3×R8 carp, which are the offspring of a cross between fish of Hungarian origin (R8 strain) and of Polish origin (R3 strain), were used (29). All experiments were performed with the approval of the animal experimental committee of Wageningen University. To immunize fish against Trypanoplasma borreli parasites, each fish was injected with 1 × 104 parasites. Two weeks later, when parasitemia reached 1 × 106–1 × 107 parasites/ml blood, fish were injected once with 10 mg/kg melarsoprol (Arsobal), a human antitrypanosome drug shown to be effective against carp trypanosomes (30). All fish survived the treatment and were kept until further use for leukocyte isolation.
Cell isolation and culture
Fish were killed with an overdose of Tricaine Methane Sulfonate (Crescent Research Chemicals, Phoenix, AZ) for organ isolation. PBLs, thymocytes, mid-kidney leukocytes, and HKLs were isolated as described previously (31, 32). Head kidney–derived mature macrophages (referred to as macrophages) were obtained upon 6 d culture of HKLs, as previously described (33).
MACS was used in combination with carp leukocyte–specific Abs to isolate neutrophils from mid-kidney leukocytes and B cells from PBLs, as previously described (30, 34–36). The purity of the sorted leukocytes was >98%, as confirmed by flow cytometry using a BD FACSCanto A (BD Biosciences). After isolation, cells were washed in RPMI 1640 with l-glutamine and 25 mM HEPES (Lonza, Nalgene) medium adjusted to 280 mOsmol/kg, supplemented with 2 mM l-glutamine, 100 U/ml penicillin G, and 50 mg/ml streptomycin sulfate (carp RPMI 1640 medium [cRPMI] with HEPES), and resuspended in the appropriate medium for the experiment, as described below.
Oxygen radical production (luminol)
To measure the production of oxygen radicals by carp neutrophils and macrophages, a real time luminol-ECL assay was performed, as previously described (37). Briefly, 50 μl luminol (10 mM; Sigma-Aldrich; in 0.2 M borate buffer [pH 9.0]) and 50 μl stimulus (0.005–0.5 U/ml rIl10 and/or 0.1 μg/ml PMA) were added to the wells of a white 96-well plate with opaque bottom (Corning). Fifty microliters of cell suspension (1 × 106 neutrophils or 0.5 × 106 macrophages/well) in cRPMI was added, and chemiluminescence emission was measured with a FilterMax F5 Multi-Mode Microplate Reader at 27°C every 3 min for 90 min.
Nitrogen radical production
The production of nitrogen radicals was determined as previously described (38). Briefly, neutrophils (1 × 106 /well) or macrophages (0.5 × 106 /well) were seeded in 96-well plates (Corning) and stimulated with 20 μg/ml LPS in combination with different concentrations of rIl10 in a total volume of 150 μl complete cRPMI (cRPMI supplemented with 1.5% pooled carp serum) or complete NMGFL-15 medium (33) (in the case of macrophages). Neutrophils were incubated for 4 d and macrophages were incubated for 24 h at 27°C in the presence of 5% CO2. After incubation, 75 μl cell culture supernatant was transferred to a new plate and combined with 100 μl 1% (v/v) sulfanilamide in 2.5% (v/v) phosphoric acid and 100 μl 0.1% (w/v) N-naphthyl-ethylenediamine in 2.5% phosphoric acid. OD540 was measured after 10 min using a FilterMax F5 Multi-Mode Microplate Reader.
Genome data mining for carp sequences
Full-length sequences for carp il10r1 (crfb7), socs3a, socs3b, zap70, rorγt, and foxp3 homologs were identified through data mining and synteny analysis of the draft carp genome [Bioproject PRJNA73579 (39)] and using previously annotated and characterized sequences (Table I). All six genes were present in duplicate and were designated as il10r1a and il10r1b, socs3aa and socs3ab, socs3ba and socs3bb, zap70a and zap70b, rorγta and rorγtb, and foxp3a and foxp3b. Common primers detecting both copies of the il10r1, socs3a, socs3b, zap70, rorγt, and foxp3 isoforms were designed (Table I) and used for gene expression analysis. Sequences were confirmed by standard PCR followed by sequencing of the products.
Gene expression analysis by real-time quantitative PCR
To measure changes in gene expression, 3 × 106 cells were stimulated with LPS (20 μg/ml) and/or 0.5 U/ml rIl10 for 3 or 6 h. The optimal time point for the expression of the proinflammatory cytokines and mhc genes was determined to be 6 h, whereas 3 h was optimal for socs3 expression. Total RNA was isolated using the RNeasy Kit (QIAGEN), including on-column DNase treatment, according to the manufacturer’s instructions, and stored at −80°C. Prior to cDNA synthesis, 1 μg total RNA was treated with DNase I, Amplification Grade (Invitrogen), and cDNA was synthesized using random primers (300 ng) and Superscript III First-Strand Synthesis for RT-PCR (Invitrogen). cDNA samples were diluted in nuclease-free water prior to real-time quantitative PCR (RT-qPCR) analysis.
RT-qPCR analysis was performed with a Rotor-Gene 6000 (Corbett Research) using ABsolute qPCR SYBR Green Mix (Thermo Scientific). The primers used for RT-qPCR are shown in Table I. Fluorescence data from RT-qPCR experiments were analyzed using Rotor-Gene Analysis software version 1.7. The take-off value for each sample and the average reaction efficiencies (E) for each primer set were obtained upon comparative quantitation analysis using Rotor Gene Software. The relative expression ratio (R) of a target gene was calculated based on the average E and the take-off deviation of sample versus control and expressed relative to the s11 protein of the 40s subunit as reference gene.
Stat3 phosphorylation
HKLs (5 × 106) were incubated with 0.25 U/ml rIl10 for 5, 15, or 30 min, pelleted, and lysed by adding 30 μl lysis buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 1 mM PMSF) and by vortexing vigorously. After 15 min of incubation on ice, lysates were centrifuged at 10,000 × g, the supernatants containing the cytoplasmic fraction were collected, and the protein concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific).
Cell lysates (20 μg) were resolved on a 10% SDS-PAGE gel and transferred at 15 V for 30 min to nitrocellulose membranes (Protran; Schleicher & Schuell BioScience) in a Trans-Blot SD Transfer Cell (Bio-Rad). The membranes were blocked for 2 h at RT with 5% (w/v) BSA in TBST (50 mM Tris, 0.15 M NaCl [pH 7.4], 0.2% [v/v] Tween-20) and incubated overnight at 4°C with a 1:1000 dilution of anti–phospho-Stat3 (Tyr705) (3E2) mouse mAb (Cell Signaling) in 5% BSA TBST. HRP-conjugated goat anti-mouse Ab (1:2000; Dako) in 5% BSA was used as secondary Ab (1 h at RT), and the proteins were visualized by chemiluminescence detection (Western Bright ECL Western blotting Detection Kit; Advansta) on x-ray films. Membranes were stripped according to standard protocols using a mild stripping buffer (1.5% glycine [w/v], 0.1% SDS, 0.1% [v/v] Tween-20 [pH 2.2]) and incubated with a 1:2000 dilution of a cross-reacting rabbit anti- human β-tubulin Ab (Abcam) as a primary Ab and a 1:2000 dilution of HRP-conjugated goat anti-rabbit Ab (Dako), according to the protocol described above.
B cell and T cell proliferation
Proliferation was monitored using CFSE labeling. Briefly, total PBLs or HKLs were washed and resuspended in 0.1% BSA in PBS, and a final concentration of 10 μM CFSE/5 × 106 cells was added. Cells were incubated at 27°C for exactly 10 min, diluted five times with cold RPMI 1640 containing 10% FBS, incubated on ice for 5 min, and washed three times with cold RPMI 1640 containing 10% FBS. After the washes, cells were recounted and resuspended in Advanced DMEM/F-12 (Life Technologies) supplemented with 2 mM l-glutamine, 100 U/ml penicillin G, 50 mg/ml streptomycin sulfate, 1% FBS, and 10−5 M 2-ME.
CFSE-labeled cells (2 × 106/well) were seeded in a 48-well plate (Corning) and stimulated with rIl10 and/or whole T. borreli lysate (0.5 parasites/cell) for 6 d at 27°C in the presence of 5% CO2. Prior to each analysis, propidium iodide (5 μg/ml) was added to a fraction of all samples to monitor cell viability. Only samples with >90% cell viability were used. Various concentrations of IL-10 (0.5, 0.25, and 0.1 U/ml) were used in preliminary experiments. A concentration of 0.25 U/ml was determined to be the lowest concentration to show optimal regulatory activities on carp leukocytes; therefore, it was used for all subsequent experiments.
For analysis of T cell proliferation, nonadherent HKLs were washed in FACS buffer (0.5% BSA, 0.05% NaN3 in PBS), fixed, and permeabilized using the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences), following the manufacturer’s instructions. Cells were then washed in FACS buffer and incubated with 50 μl a 1:100 dilution of anti-Zap70 rabbit mAb (99F2; Cell Signaling) in FACS buffer for 30 min on ice. Following two washes, 50 μl a 1:100 dilution of R-PE goat anti-rabbit IgG (H+L) Ab (Invitrogen Molecular Probes) was added, and cells were incubated for 30 min on ice before the last washes and flow cytometric analysis on a FACSCanto A (BD Biosciences).
For analysis of B cell proliferation, nonadherent PBLs or HKLs were fixed and permeabilized following the protocol described above and stained using a 1:50 dilution of WCI12 Ab [mouse monoclonal anti-carp IgM (45)] and a 1:100 dilution of polyclonal goat anti-mouse Igs/RPE, Goat F(ab′)2 (Dako) Ab. Pax5 staining was performed using a 1:100 dilution of Pax5 (D19F8)XP(R) rabbit mAb (Cell Signaling) and donkey anti-rabbit IgG-PerCP-Cy5.5 (Santa Cruz, 1:100).
Detection of total and Ag-specific IgM in cell culture supernatants
Secretion of total IgM or IgM specific for T. borreli Ags was quantified in supernatants of the same cell cultures used to study B cell proliferation. Total IgM was measured by capture ELISA; all incubation steps were performed at RT unless stated otherwise. Briefly, 96-well ELISA plates were coated with WCI12 (anti-IgM Ab) 1:500 in 100 μl bicarbonate buffer (pH 9.6) overnight at 4°C, washed, and blocked with 150 μl Universal Casein Diluent/Blocker (SDT) for 1 h. A total of 100 μl cell culture supernatant was added to each well and incubated for 1 h; after washing, biotinylated WCI12 (1:500) was added in 100 μl PBS-0.1% Tween 20, incubated for 1 h, and washed. Samples were then incubated with Streptavidin-Poly-HRP80 (SDT; 1:5000 in PBS-0.1% Tween 20) for 30 min; after careful washing, the ELISA was developed using ABTS substrate (Roche), and OD405 was measured in a FilterMax F5 Multi-Mode Microplate Reader.
A direct ELISA was performed to measure T. borreli–specific Abs. ELISA plates were coated with 2 μg parasite Ags in 100 μl bicarbonate buffer overnight at 4°C and blocked with 150 μl 0.5% BSA in TBST for 1 h. The supernatants were added and incubated for 1 h, washed, and incubated with WCI12 (1:500) in TBST for 1 h and subsequently with HRP-conjugated goat anti-mouse Ab (Dako; 1:1000 in 100 μl TBS-T) for 1 h. After careful washing, plates were developed using ABTS substrate, and the OD405 was measured in a FilterMax F5 Multi-Mode Microplate Reader.
Statistical analysis
Statistical analysis was performed using R statistical software (3.0.2) (46). The tests were performed on means or on proportions (for the proliferation assays) after testing that the data met the normality conditions using the Shapiro–Wilk test for small samples. When the conditions were not met, nonparametric tests (Mann–Whitney–Wilcoxon) were used. For normally distributed datasets, significant differences were evaluated using the Student t test. In all cases, data were analyzed as paired data to eliminate the interference caused by the high variability among individual fish. For gene-expression analysis, relative expression ratios (R) were calculated as described, and transformed [LN(R)] values were used for statistical analysis. For multiple comparisons, we used one-way ANOVA, followed by the Tukey test. In all cases, significant differences were considered at p < 0.05.
Results
Carp Il10 downregulates radical production by carp phagocytes
To investigate whether carp Il10 exerts the prototypical inhibitory activities on phagocytes, we studied the effect of rIl10 on oxygen and nitrogen radical production by carp neutrophils and macrophages. Activities were determined on PMA- or LPS-activated cells because Il10 alone had no effect on oxygen (data not shown) or nitrogen radical production. Production of oxygen radicals by neutrophils and macrophages was measured after incubation with PMA in combination with different concentrations of rIl10 (Fig. 1A). Il10 reduced the PMA-induced radical production in a dose-dependent manner. At high concentrations of Il10 (0.5 U/ml), production of oxygen radicals was almost completely inhibited in both cell types. At low concentrations of Il10 (0.005 U/ml), the inhibitory effect was negligible on neutrophils but still significant on macrophages. Preincubation for 1 h with Il10 did not increase the inhibitory effect of Il10 on oxygen radical release (data not shown). Production of nitrogen radicals by neutrophils and macrophages was measured after incubation with LPS in combination with different concentrations of rIl10 (Fig. 1B). Il10 reduced the LPS-induced nitrogen radical production in a dose-dependent manner. At high concentrations of Il10 (0.5 U/ml), radical production was significantly inhibited in both cell types. Overall, these data suggest that carp Il10 exerts prototypical inhibitory activities on fish phagocytes.
Carp Il10 inhibits radical production in carp phagocytes. (A) Respiratory burst analysis on neutrophils or macrophages stimulated with PMA (0.1 μg/ml) alone or in combination with increasing concentrations of Il10 (U/ml). The chemiluminescence units (CL Units) were measured every 3 min for 90 min. (B) NO assay performed on neutrophils or macrophages stimulated with LPS (20 μg/ml) alone or in combination with an increasing concentration of Il10 (U/ml). OD540 was measured, and the concentration was estimated using a nitrite standard curve. Bars represent mean + SD of triplicate measurements from one representative experiment out of three performed independently for each cell type. Asterisks (*) indicate significant difference relative to the respective PMA- or LPS-treated group.
Carp Il10 downregulates the expression of proinflammatory and mhc genes in carp phagocytes
In mammals, IL-10 directly downregulates the expression of proinflammatory and MHC molecules in innate immune cells, limiting the inflammatory response and indirectly regulating activation of adaptive immunity (5). To assess the direct modulatory effects of carp Il10 on gene expression, neutrophils and macrophages were stimulated for 6 h with LPS in combination with Il10, and the expression of several proinflammatory genes, as well as genes involved in Ag presentation (Table I), was measured (Fig. 2). Carp Il10 strongly inhibited the LPS-induced gene expression of il1β in both neutrophils and macrophages, of inos in neutrophils in particular, and of il6 in macrophages in particular. The inhibitory effect was less pronounced on tnfα in both cell types and on il6 in neutrophils, possibly also as a result of the low induction of these cytokines’ gene expression by LPS. No significant upregulation of ifnγ gene expression was observed in macrophages and neutrophils stimulated with LPS, whereas an interesting, although not statistically significant, upregulation of ifnγ was found in neutrophils stimulated with LPS in the presence of Il10. Of interest, Il10 also strongly inhibited the LPS-induced gene expression of the p35 subunit of il12 in macrophages and of both mhc class I (ua1) and mhc class II (dab2) in carp neutrophils. Altogether, these data suggest that Il10 downregulates the expression of proinflammatory and mhc genes in innate immune cells of fish.
Carp Il10 downregulates the expression of proinflammatory and mhc genes. Neutrophils or macrophages were stimulated for 6 h with LPS (20 μg/ml) alone or in combination with Il10 (0.5 U/ml). Gene expression was normalized relative to the s11 protein of the 40s subunit as a reference gene and is shown relative to the unstimulated controls (dashed line at y = 1). Data are mean + SD of n = 3 fish for neutrophils and n = 4 fish for macrophages. Asterisks (*) indicate significant differences from the LPS-treated samples.
Carp Il10 signals via a conserved pathway
In mammals, IL-10 signals via IL-10R1 of the IL-10R complex, activating a JAK/STAT signaling pathway leading to phosphorylated STAT3, which, upon dimerization, translocates to the nucleus where it starts the transcription of, among others, SOCS3 as an important inhibitor of proinflammatory responses (3). To verify the conservation of this pathway in fish, we studied carp il10r1 and socs3 gene expression and phospho-Stat3 levels in carp cells. Constitutive gene expression of il10r1 (crfb7) was high in carp gut, kidney, and spleen but low in skin (Fig. 3A). Although il10r1 was expressed in all leukocyte subpopulations tested, it was expressed most highly in carp macrophages (Fig. 3B), which could help to explain the pronounced inhibitory effect of Il10 on the production of oxygen and nitrogen radicals by macrophages (Fig. 1). Phospho-Stat3 levels in the cytoplasm of Il10-treated carp leukocytes increased as early as 5 min after stimulation, as shown by Western blot (Fig. 3C). Phospho-Stat3 levels remained high until 15 min after treatment with Il10, after which they declined, most probably as a result of translocation of the transcription factor to the nucleus. Gene expression of socs3, in particular socs3b, was upregulated at 3 h following treatment of carp leukocytes with Il10 (Fig. 3D). Our data suggest that the signaling pathway for Il10 could be conserved from mammals to fish.
Conservation of Il10 signaling pathway. Constitutive gene expression of il10r1 in immune organs (A) and cell types (B). Basal gene expression was normalized relative to the s11 protein of the 40s subunit as a reference gene. (C) Western blot analysis of Stat3 phosphorylation. HKLs were incubated with Il10 (0.25 U/ml) for the indicated times. Whole-cell lysates were assessed for the presence of p-Stat3 using an anti–p-Stat3 Ab. A cross-reacting anti-tubulin Ab was used as a control (52 kDa). (D) Gene-expression analysis of socs3 isoforms upon Il10 stimulation. HKLs were stimulated for 3 h with Il10 (0.25 U/ml). Gene expression was normalized relative to the s11 protein of the 40s subunit as a reference gene and is shown relative to the unstimulated controls (dashed line at y = 1). Data are mean + SD of n = 4 fish. Asterisks (*) indicate significant differences relative to the unstimulated control.
Carp Il10 promotes proliferation of memory T cells
Mammalian IL-10 inhibits both proliferation and cytokine synthesis of Th1, Th2 (directly), and Th17 cells (indirectly), inducing the development of regulatory T cells (Tregs) (5). In contrast, IL-10 can act as a growth factor to increase proliferation of specific subsets of CD8+ T cells (47). To study the effect of carp Il10 on T cell proliferation, leukocytes from naive fish and from fish that survived an infection with the parasite T. borreli (immunized) were incubated in vitro with Il10 alone or in combination with parasite Ags. Effects of Il10 on T cells were monitored by flow cytometry using Zap70 as a pan-T cell marker, whereas cell division was quantified by CFSE staining (Fig. 4). At day 0, T cells accounted for 14% of the total leukocyte population in both naive and immunized fish, with all cells being CFSE+ (data not shown). In all cell cultures from naive fish, after 6 d, the percentage of T cells decreased from 14 to ∼2% of all leukocytes in all groups (Fig. 4A, upper panels). When proliferation of the Zap70+ population was analyzed in the same cell cultures (Fig. 4A lower panels), Il10 alone did not affect proliferation of T cells, whereas parasite Ags alone induced proliferation of a small number of T cells, which could be suppressed by treatment with Il10. In contrast, in cultures from immunized fish, the percentage of T cells increased after 6 d of culture in the presence of parasite Ags, an effect that was significantly enhanced by treatment with carp Il10, whereas Il10 alone had no effect on T cell numbers (Fig. 4B, upper panels). The increase in T cell numbers observed upon stimulation with parasite Ags and Il10 could be ascribed to the strong proliferation of a small group of Ag-specific memory T cells (Fig. 4B, lower panels), because this proliferation was only observed in immunized fish.
Il10 promotes survival and proliferation of memory T cells. CFSE-labeled HKLs from naive (A) and T. borreli–immunized (B) fish were stimulated with Il10 (0.25 U/ml) and/or parasite Ags (0.5 parasites/cell) for 6 d. T cells were identified using an anti-Zap70 Ab, and their proliferation was monitored by CFSE analysis by flow cytometry. Density plots from naive and immunized fish at day 0 or after 6 d of incubation with Il10, parasite Ags, or both (upper panels). Count versus CFSE graphs of the corresponding Zap70+ populations (lower panels). The gate for undivided (Undiv) cells was set including 100% Zap70+ cells at day 0 of culture. Numbers indicate the percentage of total T cells (Zap70+, upper panels) or of dividing T cells (lower panels) ± SD of n = 3 fish. Asterisks (*) indicate significant differences between the two groups indicated by the brackets.
To investigate whether proliferation could be ascribed to a specific subset(s) of T cells in immunized fish, given the lack of suitable Abs against carp T cell markers, gene expression analysis was performed on the same cell cultures (Table II). Stimulation with Il10 alone did not lead to changes in gene expression of T cell markers (data not shown), in agreement with the absence of T cell proliferation observed in these cell cultures (Fig. 4, lower panels). In contrast, in cultures treated with parasite Ags alone or in combination with Il10, the expression of zap70 was upregulated (Table II) and correlated with the increased number of Zap70+ cells observed by flow cytometry (Fig. 4B, upper panels). Stimulation with parasite Ags alone induced an increase in cd4 expression that decreased in the presence of Il10, shifting toward a higher expression of cd8. To further characterize the proliferating Zap70+ population induced by treatment with parasite Ags, we studied the expression of transcription factors and cytokines associated with specific Th subsets. t-bet and ifnγ (Th1), as well as gata3 and il4/13 (Th2), increased upon stimulation with parasite Ags and decreased in the presence of Il10, suggesting that these subsets might be induced by specific Ag stimulation but are negatively regulated by Il10. Despite the considerable increase in gene expression of il6 (Th17) and, to a lesser extent, of il10 (Tregs), expression of rorγt (Th17) and foxp3 (Treg) remained unchanged after stimulation with parasite Ags alone, and the expression of all of these genes decreased when Il10 was used in combination with parasite Ags, suggesting that these subtypes are negatively regulated by Il10 and that the proliferating Zap70+ population does not consist primarily of Th17 or Treg subpopulations.
The gene expression data provide a first indication that the proliferating (Zap70+) T cell population in cultures from immunized fish stimulated with autologous Ags can be ascribed to the expansion of different T cell subpopulations. In cell cultures stimulated with parasite Ags alone, proliferation most likely is associated with the expansion of a subset of Th1 and Th2 memory T cells; in cell cultures stimulated with parasite Ags in combination with Il10, Il10 downregulated the proliferation of both Th1 and Th2 memory T cells and promoted proliferation of a subset of CD8+ memory T cells. Altogether, our data suggest that carp Il10 may exert differential effects on memory CD4+ and CD8+ T cells in immunized fish.
Il10 promotes IgM+ B cell proliferation
Mammalian IL-10 has a general stimulatory role on B cells, preventing apoptosis and increasing proliferation and Ig secretion (3). To study the effect of carp Il10 on B cell proliferation, leukocytes from naive and T. borreli–immunized fish were incubated in vitro with Il10 alone or in combination with parasite Ags. Effects of Il10 on B cells from HKLs and from PBLs were monitored by flow cytometry using an anti-carp IgM Ab (WCI12) as a pan-IgM+ B cell marker, whereas cell division was quantified by CSFE staining (Fig. 5). In HKL-derived cell cultures, IgM+ B cell proliferation was especially high in cultures from both naive (Fig. 5A, 64.9–84.4%) and immunized (Fig. 5B, 65.3–73.4%) fish, either unstimulated or treated with Il10 alone. Treatment with parasite Ags in combination with Il10 did not lead to a significant additional increase in IgM+ B cell proliferation. In PBL-derived cell cultures, IgM+ B cell proliferation was generally low in unstimulated cultures from both naive (Fig. 5C, 11.8%) and immunized (Fig. 5D, 19.3%) fish, but it clearly increased when cells were stimulated with parasite Ags (Fig. 5C, 34.5%; Fig. 5D, 47.8%) or Il10 alone (Fig. 5C, 44.8%; Fig. 5D, 58.2%). Treatment with parasite Ags in combination with Il10 strongly increased the proliferation of peripheral IgM+ B cells from naive (Fig. 5C, 57.6%) and immunized (Fig. 5D, 61.1%) fish. Our findings indicate that carp Il10 has a general stimulatory role on fish IgM+ B cells by increasing proliferation.
Il10 promotes proliferation of IgM+ B cell from PBLs. Proliferation of the IgM+ B cell population was monitored by CFSE analysis of labeled HKLs (A and B) and PBLs (C and D) from naive and T. borreli–immunized fish stimulated with Il10 (0.25 U/ml) and/or parasite Ags (0.5 parasites/cell) for 6 d. Total IgM+ B cells were stained using the WCI12 Ab, and the gate for undivided (Undiv) cells was set including 100% IgM+ cells at day 0 of culture. Numbers represent the mean percentage of dividing B cells ± SD of n = 3 fish. Asterisks (*) indicate significant differences between the two groups indicated by the brackets.
Il10 increases total and specific IgM secretion
Mammalian IL-10 is known to stimulate B cell proliferation, as well as to increase Ab secretion. Therefore, given the stimulatory effect of carp Il10 on IgM+ B cell proliferation, we next investigated whether this resulted in the regulation of total or Ag-specific IgM secretion in cell culture supernatants from naive and immunized fish. The effect of Il10 on total IgM secretion was measured in the supernatants of HKL- and PBL-derived cell cultures stimulated with Il10 alone using a capture ELISA.
In HKL-derived cell cultures, treatment with Il10 significantly increased the concentration of secreted total IgM, but only in supernatants derived from immunized fish (Table III), despite the lack of significant effects of Il10 on IgM+ B cell proliferation under the same conditions (Fig. 5A, 5B). In contrast, in PBL-derived cell cultures, no significant differences compared with the unstimulated control were observed with regard to secreted total IgM in naive or immunized fish (Table III), despite the stimulatory effect of Il10 on B cell proliferation observed under the same conditions (Fig. 5C, 5D).
The increase in total IgM secretion in supernatants from HKL-derived cell cultures from immunized fish, in particular, suggested that Il10 might exert an effect on Ag-specific B cells and prompted us to measure T. borreli Ag–specific IgM in the same supernatants (Table IV). Again, in supernatants from HKL-derived cell cultures from immunized fish, treatment with Il10 in combination with T. borreli Ags increased the concentration of secreted Ag-specific IgM with respect to cultures treated with Il10 or T. borreli Ags alone. These findings indicate that carp Il10 increases total and Ag-specific IgM secretion by fish B cells.
Il10 promotes IgM+ B cell differentiation
Carp Il10 has a stimulatory role on fish IgM+ B cells by increasing proliferation in PBL-derived, but not HKL-derived, cell cultures (Fig. 5), whereas treatment with carp Il10 increased IgM secretion from HKL-derived, but not PBL-derived, B cell cultures (Tables III, IV). This apparent contrast suggested that the IgM+ B cell population in PBLs and HKLs is heterogeneous and that carp Il10 might have differential effects on the various developmental stages of IgM+ B cells or affect their differentiation. Therefore, the effects of Il10 on different B cell developmental stages were monitored by flow cytometry using the anti-carp IgM Ab (WCI12), in combination with an Ab against the transcription factor Pax5, as a marker for B cell differentiation (Fig. 6), adhering to the previously defined nomenclature for developmental stages of fish B cells (48).
Il10 promotes IgM+ B cell differentiation. HKLs (A) and PBLs (B) from immunized fish were stimulated with Il10 (0.25 U/ml) and/or parasite Ags (0.5 parasites/cell) for 6 d and stained with anti-IgM and anti-Pax5 Abs to study B cell–differentiation stages. In (A), the gating was performed independently for days 0 and 6 as a result of differences in the staining intensity. Shown is the IgM+ population; the gating was performed to separate IgMhigh and IgMlow and Pax5high and Pax5low populations. The names used to identify the populations follow the nomenclature of Zwollo et al. (48).
In HKL-derived cell cultures from immunized fish at time point 0, three differentiation stages of B cells were identified based on their IgM and Pax5 levels: developing B cells (dBs), plasmablasts (PBs), and PCs (Fig. 6A). In vitro culture of HKLs for 6 d induced IgM+ B cell differentiation, leading to an increase in the PC population. Treatment with Il10 alone induced a further differentiation from PB to PC populations, supporting the previously observed increase in secreted total IgM in the supernatants of these cell cultures (Table III). Treatment with parasite Ags did not induce changes compared with the control samples; however, in combination with Il10, an increase in differentiation from dBs to PBs and, subsequently, to PCs was observed, supporting the previously observed increase in secreted Ag-specific IgM in the supernatants of these cell cultures (Table IV). Triple staining for IgM, Pax5, and CFSE (data not shown) confirmed that the increase in proliferation observed in all HKL cultures, either stimulated or not (Fig. 5B), could be ascribed to an overall proliferation and differentiation of all IgM+ B cell stages identified in the HKL cultures, each in different proportions, depending on the stimulus. These data show a clear effect of carp Il10 on the maturation and differentiation of HK-derived IgM+ B cells into PCs.
In PBL-derived cell cultures from immunized fish at time point 0 (Fig. 6B), only a single differentiation stage of IgM+ B cells was identified: mBs. In vitro culture of PBLs for 6 d induced IgM+ B cell differentiation, leading to the appearance of the PB population. Treatment with Il10 alone induced proliferation of the mB population, whereas treatment with parasite Ag, especially in the presence of Il10, induced an even stronger proliferation of mBs. Triple staining for IgM, Pax5, and CFSE (data not shown) confirmed that the increase in proliferation observed in all PBL-treated cultures (Fig. 5D) could be ascribed to the differentiation of mBs into PBs (especially in the unstimulated control), as well as proliferation of mBs in all treated cultures. Furthermore, the lack of PCs in all PBL-derived cell cultures supports the limited changes in total or Ag-specific secreted IgM measured in these culture supernatants (Tables III and IV).
Altogether, these data suggest that Il10 supports the overall proliferation, differentiation, and Ab secretion of IgM+ B cells in carp. In PBLs, Il10 supports the proliferation of mBs and their differentiation into PBs, which are unable, however, to secrete high levels of Abs. In head kidney (bone marrow equivalent), Il10 supports the generation of a large pool of (polyclonal and Ag-specific) PCs that is able to secrete Abs.
Discussion
In the current study, we investigated the effects of carp Il10 on phagocytes and lymphocytes. We show that Il10 shares several prototypical activities with mammalian IL-10, including the ability to downregulate the inflammatory response of phagocytes, support proliferation of specific subsets of memory T lymphocytes, and stimulate differentiation and Ab secretion by IgM+ B lymphocytes. To our knowledge, this is the first comprehensive analysis of biological activities of fish Il10 that includes effects on both phagocytes and lymphocytes. Our results point to a strong conservation of Il10 functions.
Deactivation of phagocytes, in particular of macrophages, is among the most typical activities of IL-10 (49–52). In the first part of this study, we analyzed the biological activities of carp Il10 on both macrophages and neutrophils, focusing on the ability of Il10 to modulate radical production and proinflammatory gene expression. Our results show that carp Il10, when added before (data not shown) or at the same time as the stimulus (Fig. 1), downregulated the PMA-induced oxygen radical production and the LPS-induced nitrogen radical production by both macrophages and neutrophils. In general, the inhibitory effects of Il10 were more pronounced in carp macrophages than in neutrophils. Human macrophages display high surface levels of IL-10R1, whereas neutrophils store IL-10R intracellularly and require stimulation with LPS, TNF-α, or GM-CSF to promote surface display of the receptor. In humans, this explains a difference between the responsiveness of macrophages and neutrophils to the inhibitory effects of IL-10, at least with regard to oxygen radical production (53). Differences in Il10R surface display between macrophages and neutrophils could help to explain our data but have not been studied in carp. The less pronounced inhibitory effect of Il10 on nitrogen radical production by carp neutrophils also can be explained by the relatively long incubation time required to measure accumulation of nitrite in the supernatant of neutrophils. It is likely that the inhibiting effects of Il10 are transient [as also shown in mammals (50)] and occur during the first hours of incubation, after which neutrophils can restore the production of NO in the presence of LPS, thereby leading to less marked differences between Il10-treated and nontreated cells. Other studies in mammals showed that IL-10 inhibits PMA-triggered oxygen radical production and, to a lesser extent, IFN-γ–triggered NO production in macrophages when added before or at the same time as the stimulus (49). In mammals, IL-10–mediated deactivation of phagocytes is, to a large degree, attributed to an indirect effect of IL-10, which, when added prior to the stimulus, downregulates TNFα expression, thereby leading to reduced radical production (50, 54). This indirect route may not necessarily apply to our findings, because the role of TNF-α as a prototypical proinflammatory molecule in teleost fish is still controversial: in trout and goldfish, for example, a more conserved function of the stimulatory activities of TNF-α was described (55, 56), whereas in sea bream (57) and carp (58), TNF-α was unable to exert prototypical proinflammatory activities on phagocytes. Therefore, based on our current knowledge (58), it seems unlikely that, at least in carp, TNF-α is involved in the PMA- or LPS-mediated induction of radicals. Furthermore, our gene expression analysis showed that, within a short time span of 6 h, the addition of Il10 inhibited inos expression, suggesting the presence of a rapid, direct inhibitory effect of Il10 on nitrogen radical production in both neutrophils and macrophages. In addition, the downregulation of proinflammatory molecules, such as il1β or il12 (p35), was already observed at 6 h. Last, but not least, the inhibitory effect of Il10 on oxygen radical production occurred so rapidly (15 min, Fig. 1A) that a direct, rather than an indirect, pathway is most likely responsible for the deactivating effects of Il10 on carp phagocytes.
Inhibition of Ag presentation by both MHC class I and MHC class II is another well-known activity of IL-10 (59, 60). Carp Il10 induced downregulation of mhc-I and mhc-II genes in neutrophils, but no clear effect on mhc gene expression was observed in macrophages, at least at the time point analyzed. In human monocytes, IL-10 induces an intracellular accumulation of mature MHC class II complexes, preventing their display at the plasma membrane, rather than having an effect on MHC-II gene transcription or protein synthesis (61). Thus, it is likely that gene expression analysis alone might not be sufficient to fully detect inhibition of Ag presentation induced by carp Il10 in phagocytes; a further analysis of cellular localization of MHC proteins after Il10 treatment would be required to determine whether inhibition of Ag presentation is a common feature of carp Il10. Analysis of gene expression confirmed the ability of carp Il10 to downregulate the expression of several proinflammatory genes and upregulate the expression of socs3 genes, which, as proteins, are involved in the negative regulation of proinflammatory cytokines (62). Anti-inflammatory activities of Il10 also were described for goldfish monocytes: preincubation with goldfish Il10 downregulated the expression of proinflammatory cytokines, including tnfα, ifnγ, and cxcl8, and Il10 deactivated monocytes by reducing oxygen radical production induced by bacteria and IFN-γ (23). Interestingly, in the same study, Il10 was shown to directly decrease the expression of the NADPH oxidase component p47phox in activated monocytes, supporting the suggestion that a direct effect of Il10 on fish phagocytes might be responsible for phagocyte deactivation. Similar to what we showed in this study, goldfish Il10 induced phosphorylation of Stat3 and increased soscs3 gene expression (23). Considering the results in goldfish and carp together, it seems clear that fish Il10 exerts prototypical anti-inflammatory activities on monocytes, macrophages, and neutrophils, most likely through a conserved signaling pathway.
Direct and indirect effects of IL-10 on T cell functions have been described extensively in mammals (reviewed in Refs. 3, 63). Therefore, in the second part of the study, we focused on the regulatory activities of carp Il10 on T cells, in particular the ability of Il10 to regulate lymphocyte proliferation and effector functions, such as cytokine expression. In mammals, the effects of IL-10 on T cells can be indirect, via the regulation of the production of cytokines, or direct, via IL-10R (5, 64). Indeed, general inhibitory effects of IL-10 on CD4+ T cells largely can be traced back to the ability of IL-10 to inhibit Ag presentation and cytokine production by APCs and, thus, should be considered an indirect effect of IL-10 on T cells (4, 49, 65–68). In addition to these indirect effects on T cells via inhibition of APC function, IL-10 can have direct effects on T cells, depending on the subtype and activation state. For example, IL-10 shows inhibitory activities toward resting CD4+ T cells via the suppression of IL-2, IFN-γ, IL-4, and IL-5 production (69–71) or via inhibition of the CD28 signaling pathway (reviewed in Refs. 3, 72). Direct effects of IL-10 on CD8+ T cells also were observed, although apparently contradictory results were reported. For example, IL-10 could stimulate as a growth factor the proliferation of certain subsets of purified CD8+ T cells upon mitogen stimulation or when administered in combination with IL-2 (47, 73, 74), whereas in another study, IL-10 inhibited proliferation and effector function of CD8+ T cells (75, 76). No matter what, it is clear that regulatory activities of IL-10 on T cell functions are disparate and can be time, dose, and Ag dependent.
In our study, we used head kidney (bone marrow equivalent) cell cultures from either naive or immunized animals to investigate the effects of carp Il10 on T cell (Zap70+) proliferation. Our results show that Il10 alone does not induce T cell proliferation in cultures from naive or immunized fish. This finding suggests that, similar to mammalian IL-10, fish Il10, when used alone, is not a general T cell growth factor and is not sufficient to support T cell proliferation. Interestingly, when cell cultures from immunized animals were re-exposed to parasite Ags, an increase in T cell numbers and an upregulation of cd4 and zap70 gene expression were observed. Additional gene expression analysis suggested that re-exposure to parasite Ags might induce proliferation and activation of Th1 (t-bet and ifnγ) and Th2 (gata3 and il4/13) cells. Furthermore, when cell cultures from immunized animals were restimulated with parasite Ags in combination with Il10, a further increase in T cell number was observed. This increase coincided with an upregulation of cd8 and zap70 gene expression and concomitant downregulation of cd4 and the above-mentioned Th1 and Th2 markers. Our preliminary gene expression analysis suggests that the proliferating T cell population observed in cultures stimulated with parasite Ags alone is a different T cell population from the one observed in cultures stimulated with parasite Ags in combination with Il10. In the latter, the increase in T cell proliferation was most likely due to an active proliferation of a subset of memory CD8+ T cells rather than to a mere survival of the CD8+ T cell population, because T cell proliferation and upregulation of cd8 expression were not observed in cultures from naive animals. Positive regulation of Ag-stimulated CD8+ T cells by IL-10 was described for mammalian IL-10 (75), although it was shown to be stimulus and dose dependent (77, 78). Clearly, the exact effect of IL-10 on different T cell populations is determined by the type of response, stimulus, and dose of IL-10. Our results in carp suggest that, under our experimental conditions, Il10 can inhibit Th1 and Th2 responses after autologous Ag stimulation and stimulate proliferation of memory CD8+ T cells when given at the same time as the Ag. Future studies using additional tools, such as specific Abs to CD markers, which are not available for carp, should help to positively identify subsets of T cells and, most of all, characterize different T cell (sub)populations influenced by Il10. Nevertheless, to our knowledge, our study provides the first clear indication of a regulatory role for Il10 on fish T cells.
IL-10–mediated increases in B cell survival, proliferation, terminal differentiation, and Ab secretion were reported in a multitude of studies in mammals (79–81). Therefore, in the last part of this study we focused on the regulatory activities of carp Il10 on B lymphocytes (IgM+), in particular on the ability of Il10 to regulate B cell proliferation, differentiation, and Ab production. To this end, we used PBL and HKL cultures from either naive or immunized animals to investigate the effects of Il10 on B cell activation in an Ag-driven or Ag-independent manner. In head kidney cell cultures from either naive or immunized fish, a generally high B cell proliferation was observed, and Il10 did not have enhancing effects when used alone or in combination with the immunizing Ag. Nevertheless, in head kidney cell cultures from immunized, but not from naive, fish, Il10 promoted the secretion of total and of parasite-specific IgM. Effects of Il10 were different when studied in the context of PBL cell cultures. In cell cultures from PBLs isolated from both naive and immunized fish, a clear effect of Il10 on B cell proliferation was observed when used alone or in combination with the immunizing Ag. Despite the clear effect of Il10 on B cell proliferation in these cell cultures, Il10 did not enhance the secretion of total or parasite-specific IgM. This apparent contradiction with respect to the effect of Il10 on B cell proliferation and Ab production prompted us to analyze the B cell developmental stages present in HKL and PBL cell cultures.
In trout, dBs show membrane IgM expression and high expression of the transcription factor Pax5 (48), whereas mBs can be found in the circulation and are characterized by an even higher expression of membrane IgM and intracellular Pax5. Upon differentiation into PBs and, subsequently, into PCs, expression of the transcription factor Blimp1 gradually increases, downregulating the expression of Pax5 and upregulating the expression of secreted IgM. These terminal differentiation states no longer express surface IgM but are able to secrete large amounts of Abs (48). Indeed, double staining for the presence of IgM and Pax5 showed a clear difference in the developmental B cell stages derived from either HKLs or PBLs. In head kidney cultures, Il10 greatly promoted the proliferation of dBs, PBs, and PCs, as well as the differentiation of PBs into PCs, providing an explanation for the general B cell proliferation observed in these cell cultures and the increased IgM secretion. However, in PBL cultures, Il10 greatly promoted the proliferation and differentiation of mBs and PBs but failed to induce differentiation into PCs, possibly explaining the proliferation observed in PBL cultures and the lack of secreted IgM in these culture supernatants. Terminal B cell differentiation into PCs does not seem to occur in fish PBL cultures in vitro (82). Overall, our results in carp confirm that fish Il10 might also play a pivotal role in the regulation of B cell functions.
In summary, we confirm that fish Il10 retains classical anti-inflammatory activities by inhibiting the production of reactive radicals and expression of proinflammatory genes in phagocytes. To our knowledge, this is the first report of a role for fish Il10 in the regulation of T and B lymphocytes and that Il10 affects memory T cell responses, as well as B cell proliferation, differentiation, and Ab production. Our comprehensive analysis of the biological activities of fish Il10 clearly points to an overall functional conservation of this important regulatory cytokine in teleost fish.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank Marleen Scheer, Trudi Hermsen, and Ben Meijer from the Cell Biology and Immunology group for technical support and Dr. Patty Zwollo (College of William and Mary, Williamsburg, VA) for discussions and helpful advice on the B cell experiments.
Footnotes
This work was supported by the European Community’s Seventh Framework Programme (FP7/2007-2013) under Grants PIEF-GA-2011-302444 FISHIL10 and TARGETFISH (311993), as well as by the Netherlands Organisation for Scientific Research under Veni Project 11200.
Abbreviations used in this article:
- cRPMI
- carp RPMI 1640 medium
- dB
- developing B cell
- HKL
- head kidney leukocyte
- mB
- mature B cell
- PB
- plasmablast
- PC
- plasma cell
- RT
- room temperature
- RT-qPCR
- real-time quantitative PCR
- Treg
- regulatory T cell.
- Received August 13, 2014.
- Accepted October 23, 2014.
- Copyright © 2014 by The American Association of Immunologists, Inc.
References
In this issue
