Identification of a potential common ancestor for mammalian cross-presenting dendritic cells in teleost respiratory surfaces

January 2018 | Volume 9 | Article 591 Original research published: 25 January 2018 doi: 10.3389/fimmu.2018.00059 Frontiers in Immunology | www.frontiersin.org Edited by: Lluis Tort, Universitat Autònoma de Barcelona, Spain Reviewed by: Jian-zhong Shao, Zhejiang University, China Yong-An Zhang, Institute of Hydrobiology (CAS), China *Correspondence: Carolina Tafalla [email protected]; Aitor G. Granja aitor[email protected] †These authors have contributed equally to this work. Specialty section: This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology Received: 21September2017 Accepted: 09January2018 Published: 25January2018 Citation: SoletoI, FischerU, TafallaC and GranjaAG (2018) Identification of a Potential Common Ancestor for Mammalian Cross-Presenting Dendritic Cells in Teleost Respiratory Surfaces. Front. Immunol. 9:59. doi: 10.3389/fimmu.2018.00059 identification of a Potential common ancestor for Mammalian cross-Presenting Dendritic cells in Teleost respiratory surfaces Irene Soleto1, Uwe Fischer2, Carolina Tafalla1*† and Aitor G. Granja1*† 1 Centro de Investigación en Sanidad Animal (CISA), INIA, Valdeolmos, Spain, 2 Bundesforschungsinstitut für Tiergesundheit, Friedrich-Loeffler-Institut (FLI), Insel Riems, Germany Dendritic cells (DCs) are highly specialized antigen-presenting cells that bridge innate and adaptive immune responses in vertebrates, being key modulators in the initiation of specific responses. Although teleost fish present the main elements of a fully developed adaptive immune system, not many studies have focused on identifying specific DC subsets in teleost species. Previous work from our group identified in rainbow trout (Oncorhynchus mykiss) skin a DC subpopulation co-expressing CD8α and major histocompatibility complex II β on the cell surface. Interestingly, these CD8+ DCs expressed common unique markers of mammalian cross-presenting DCs, a DC subset with an important role in antigen presentation and activation of CD8+ T cytotoxic lymphocytes. In this study, we have identified a similar DC subset in rainbow trout gills that also transcribes molecules uniquely expressed on diverse mammalian cross-presenting DC populations such as CD8, CD103, CD141, Batf3, IFN regulatory protein 8, and toll-like receptor 3. Hence, we have undertaken a broad phenotypic and functional characterization of this new DC subset that includes the confirmation of novel capacities for DCs in teleost, such an IgM-binding capacity and responsiveness to CD40 ligand. Furthermore, our results show that in gills, this DC subset shows some different phenotypic and functional characteristics when compared with their homologs in the skin, suggesting an adaptation of the cells to different mucosal tissues or different maturation status depending on their location. Our findings contribute to increase our knowledge on fish cross-presenting DCs, an important cell population to take into account for the future design of mucosal vaccination strategies. Keywords: dendritic cells, rainbow trout, gills, respiratory surfaces, cross-presentation, cD8, major histocompatibility complex ii inTrODUcTiOn Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a key role orchestrating adaptive immune responses. DCs are found in most tissues but are particularly abundant in mucosal interfaces to induce specific immunity against invading pathogens and maintain tolerance to selfor innocuous antigens (1). DCs express a wide range of pattern recognition receptors including toll-like receptors (TLRs) that allow them to sense foreign antigens. Upon sensing, they have 2 Soleto et al. Cross-Presenting DCs in Teleost Gills Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 59 the capacity to internalize antigens, process them and present them in the context of major histocompatibility complex (MHC) I or MHC II to T cells (2). Unlike other phagocytes such as macrophages or neutrophils, DCs are not involved in pathogen clearance. Thus, phagosomal degradation and acidification are much lower in DCs than in other phagocytes, and consequently the antigen is best preserved and a wider range of peptides are presented in MHC I and MHC II molecules, being this the reason for the increased capacity of DCs to prime and stimulate Tcells (3). Although teleost fish constitute the first animal group in which all the main elements of a complete adaptive immune system are present, only a few studies have addressed the characterization of DCs in fish. In salmonids, several studies suggested the presence of DCs such as those describing cells containing Birbeck granules (4), cells with DC-like morphology (5) or cells expressing transcripts of DC markers such as CD208/LAMP3 (6). Later on, a mammalian protocol was adapted in rainbow trout (Oncorhynchus mykiss) to obtain hematopoietic cultures enriched in cells that shared many DC characteristic features and showed a greater capacity than Bcells or macrophages to stimulate T cell proliferation (7). In zebrafish (Danio rerio), a leukocyte subpopulation enriched from lymphoid tissues by affinity to the lectin peanut agglutinin was also shown to share morphological and functional features of mammalian DCs (8). Also in zebrafish, CD209/DC-SIGN expression defined a cell type that co-expressed MHC II, CD80/86, and CD83 and was able to initiate antigen-specific CD4+ Tcell activation (9). All these studies confirmed the presence of DCs in teleost, but it was not until 2015 that a specific DC subset was identified in teleost fish (10). This specific subpopulation identified in rainbow trout skin consisted in large granular cells defined by high surface expression levels of MHC II and surface expression of CD8α. These cells contained negligible amounts of T cell transcripts (CD3, TCRα, and TCRβ); showed high transcription levels of characteristic DC markers such as DC-SIGN or LAMP3; were phagocytic for both polystyrene beads and apoptotic cells; were responsive to TLR ligands and to invivo stimulation; and presented a semi-mature profile with high levels of CCR7 surface expression and intermediate levels of CD83 and co-stimulatory molecules (10). Interestingly, these CD8+ DCs found in rainbow trout skin expressed common unique markers of mammalian cross-presenting DCs, strongly supporting the hypothesis of them being a common ancestor for vertebrate cross-presenting DCs. Generally, intracellular antigens (generated by virus infection or tumor development) are presented to cytotoxic CD8+ Tcells in the context of MHC class I molecules, whereas extracellular antigens are taken up by APCs that present them in an MHC II context to helper CD4+ Tcells. In addition, specific subsets of DCs, even when not infected, are able to acquire extracellular antigens, process them, and present them in the context of MHC I through a process designated as cross-presentation (11). These cross-presenting DCs are more efficient than other DC subsets in triggering effective T cytotoxic responses against intracellular pathogens and tumors (12). In mice, cross-presenting DCs include resident CD8+ DCs found in spleen, lymph nodes (LNs), and thymus (13) and migratory CD103+ DCs derived from tissues such as skin, lung, and intestine (14). In humans, a CD141hi DC subset identified in blood (15) and tissues such as dermis, and liver and lung (16) was found to have a superior capacity to cross-present than other human DC populations. Interestingly, all these cross-presenting DC populations from mice and human share common exclusive features, which are not found in other DC subsets, thus suggesting the existence of a common ancestor. For instance, all cross-presenting populations use the CLEC9A lectin to recognize necrotic cells (16–18) and express the chemokine receptor XCR1 (19) and TLR3 to respond to viral stimuli (16, 20, 21). Furthermore, the functionality of all these cross-presenting populations is regulated by the fms-like tyrosine kinase 3 ligand, IFN regulatory protein 8 (IRF8) (22, 23), and Batf3 (24, 25). Remarkably, the identification of a subpopulation of DCs in teleost skin expressing CD8, CD103, CD141, TLR3, IRF8, and Batf3 strongly pointed to these cells as a potential common ancestor for mammalian cross-presenting DCs (10). Because cross-presenting DCs had also been identified in human lungs, in the current work, we explored whether a CD8+ DC subset similar to that found in rainbow trout skin could also be identified in teleost gills, an equivalent respiratory organ. Lungs and gills are specialized respiratory surfaces that have evolved in different organisms in a quite specific manner depending on whether oxygen had to be taken up from the air or the water, their behavioral activities or their phylogenetic level of development (26). Despite these anatomical differences, all respiratory surfaces contain a specialized associated immune system that constitutes a first line of defense against airor waterborne infectious agents. In this context, our study reports the identification of a specific DC subset for the first time in teleost gills. Similarly to their skin counterpart, these gill CD8+ DCs were capable of undertaking DC-specific activities and also expressed specific markers of different mammalian crosspresenting DC subsets. In addition, our studies have revealed novel capacities for DCs in teleost, such as an IgM-binding capacity and responsiveness to CD40 ligand (CD40L). MaTerials anD MeThODs experimental Fish Female rainbow trout (O. mykiss) of ~50g were obtained from Piscifactoría Cienfuentes (Guadalajara, Spain) and maintained at the animal facilities of the Centro de Investigación en Sanidad Animal (CISA-INIA) in an aerated recirculating water system at 16°C, with 12:12h light/dark photoperiod. Fish were fed twice a day with a commercial diet (Skretting). Before any experimental procedure, fish were acclimatized to laboratory conditions for at least 2weeks. All of the experiments described comply with the Guidelines of the European Union Council (2010/63/EU) for the use of laboratory animals and have been approved by the Instituto Nacional de Investigación Agraria y Alimentaria (INIA) Ethics Committee (ORCEEA 2016-021). Tissue sampling Rainbow trout were killed by benzocaine (Sigma) overdose and gills and spleen collected. Single cell suspensions were obtained 3 Soleto et al. Cross-Presenting DCs in Teleost Gills Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 59 using 100µm nylon cell strainers (BD Biosciences) and Leibovitz medium (L-15, Invitrogen) supplemented with 100IU/ml penicillin and 100µg/ml streptomycin (P/S, Life Technologies), 10U/ml heparin (Sigma), and 5% fetal calf serum (FCS, Life Technologies). Cell suspensions were placed onto 30/51% discontinuous Percoll (GE Healthcare) density gradients and centrifuged at 500×g for 30min at 4°C. The interface cells were collected and washed twice in L-15 containing 5% FCS. Flow cytometry For the identification of DC populations, leukocytes were incubated for 30min with anti-trout CD8α (mAb rat IgG2; 7µg/ml) (27) and anti-trout MHC II [mAb mouse IgG1 coupled to allophycocyanin; 2µg/ml] (10) antibodies in L-15 media supplemented with 5% FCS. Cells were then washed twice with culture media and stained for 20min with a secondary Ab for anti-CD8α [R-phycoerythrin F(ab′)2 fragment of goat anti-rat IgG (H+L) (Life Technologies)] in L-15 media supplemented with 5% FCS. After incubation, cells were washed two times with L-15 with 5% FCS and analyzed on a FACSCalibur flow cytometer (BD Biosciences) equipped with CellQuest Pro software. To determine the levels of expression of surface CCR7 in CD8+ DC populations, the anti-trout CD8α and anti-trout MHC II (allophycocyanin-labeled) antibodies were combined with a specific anti-CCR7 polyclonal antibody (pAb rabbit IgG; 2µg/ml) (28). After 30min, the cells were washed twice with culture media and stained 20min with secondary antibodies that included an R-phycoerythrin F(ab′)2 fragment of goat anti-rat IgG (H+L) and an Alexa Fluor® 488 F(ab′)2 fragment of goat anti-rabbit IgG (H+L) (Life Technologies). After incubation, cells were washed two times with L-15 with 5% FCS and analyzed on a FACSCalibur flow cytometer. In all cases, isotype controls for mouse mAbs, rat mAb, and rabbit pAb (BD Biosciences) were tested in parallel to discard unspecific binding of the Abs, and cells were stained with propidium iodide (PI; 0.5µg/ml) to check cell viability. Flow cytometry analysis was performed with FlowJo 10 (TreeStar). confocal Microscopy Rainbow trout gills from anesthetized and exsanguinated rainbow trout were embedded in PolyFreeze cryostat mounting medium (Sigma), immediately frozen in liquid nitrogen, and stored at −80○C until used. Cryostat sections with a thickness of 14µm were prepared using a Leica CM3050 microtome and placed on SuperFrost glass slides (Menzel-Gläser). Dry sections were fixed in acetone at −20°C for 20min, air dried, encircled with a hydrophobic compound (ImmunoPen; Calbiochem), incubated for 1h at room temperature with a blocking solution (TBS buffer pH 7.5, containing 0.01% BSA, 0.5% saponin, 0.02% Tween-20, and 5% goat serum), and stained with Abs against trout MHC II β-chain (allophycocyanin-labeled) and trout CD8α. Samples were washed and incubated with Alexa Fluor 488 F(ab′)2 fragment of goat anti-rat igG (H+L) (Life Technologies). Samples were counterstained with 1µg/ml DAPI (Sigma). Laser scanning confocal microscopy three-dimensional image stacks (12 µm total thickness) were acquired with an inverted Zeiss Axiovert LSM 880 microscope. Images were analyzed with Zen 2.0 (Carl Zeiss) and Fiji (NIH) software packages. Transcriptional analysis of Facs isolated Populations Gill CD8+ DC populations were isolated by flow cytometry in a BD FACSAria III cell sorter (BD Biosciences) after staining gill leukocytes with anti-trout CD8α and anti-trout MHC II antibodies as described earlier and using their FSC/SSC and fluorescence characteristics. Splenic IgM+ Bcells and CD8+ cytotoxic Tcells (lymphoid CD8+MHC II− cells) were also isolated by flow cytometry in a BD FACSAria III cell sorter to be used in some specific experiments. For this, spleen leukocytes were incubated with anti-trout IgM mAb (1.14, mouse IgG1) coupled to APC or with a combination of anti-trout CD8α and anti-trout MHC II antibodies as previously described. RTS11, a rainbow trout monocyte/macrophage cell line established from spleen (29) was also included in some transcriptional analysis for comparative purposes. Total cellular RNA was isolated from cell populations using the Power SYBR Green Cells-to-Ct Kit (Invitrogen) following the manufacturer’s instructions. RNA was treated with DNase during the process to remove genomic DNA that might interfere with the PCR reactions. Reverse transcription was also performed using the Power SYBR Green Cells-to-Ct Kit (Invitrogen) following the manufacturer’s instructions. To evaluate the levels of transcription of the different genes, real-time PCR was performed with a LightCycler® 96 System instrument (Roche) using SYBR Green PCR core Reagents (Applied Biosystems) and specific primers (Table S1 in Supplementary Material). Each sample was measured in duplicate under the following conditions: 10min at 95°C, followed by 40 amplification cycles (15s at 95°C and 1min at 60°C). A melting curve for each primer set was obtained by reading fluorescence every degree between 60 and 95°C to ensure only a single product had been amplified. The expression of individual genes was normalized to the relative expression of trout housekeeping gene elongation factor 1α (EF-1α), and the expression levels were calculated using the 2−ΔCt method, where ΔCt is determined by subtracting the EF-1α value from the target Ct. No template negative controls and minus reverse transcriptase controls were included in all the experiments. Phagocytic activity To analyze the phagocytic capacity of gill CD8+ DCs, gill leukocytes were seeded in 24-well plates (Nunc) at a cell density of 1×106 cells/well and incubated for 16h at 20°C with fluorescent beads (FluoSpheres® Microspheres, 1.0µm, Crimson Red Fluorescent 625/645, 2% solids; Life Technologies) at a cell:bead ratio of 1:10 or without beads in the case of negative controls. After the incubation period, cells were harvested by gently pipetting, and non-ingested beads were removed by centrifugation (100 ×g for 10min at 4°C) over a cushion of 3% (weight/volume) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5% (weight/volume) d-glucose (Sigma). Cells were resuspended in L-15 with 5% FCS, labeled with the flow cytometry antibodies and analyzed on a FACSCalibur flow cytometer or under the confocal 4 Soleto et al. Cross-Presenting DCs in Teleost Gills Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 59 microscope. In some experiments, cytochalasin B (0.05µg/ml) was added to the cells immediately before the addition of the beads to verify active phagocytosis. Mixed leukocyte reaction (Mlr) Gill CD8+ DCs were isolated by cell sorting as described earlier and cultured for 12h in L-15 medium supplemented with 20% FCS in the presence of the Tcell-dependent antigen TNP hapten conjugated to the keyhole limpet hemocyanin (TNP-KLH) (Biosearch Technologies). Because no antibodies are available against extracellular pan-Tcell markers in rainbow trout, we used Tcell-enriched cultures as responder cells. These Tcell-enriched cultures were obtained from isogeneic splenocytes by depleting all IgD+, IgM+, and MHC II+ cells through cell sorting. The resulting negative population, representing approximately 10% of splenocytes, was then labeled with Cell Trace™ CFSE Cell Proliferation Kit (ThermoFisher Scientific). The enrichment in Tcells was verified by stimulation with Concanavalin A (ConA, 4µg/ml; Sigma), a typical Tcell mitogen. To carry out the MLR, stimulated DCs were cocultured with isogeneic Tcell-enriched splenocytes at a ratio of 1:30 (DCs:splenocytes). After 5days of incubation at 20°C, cocultured samples were stained with 7-AAD (BD Biosciences) at 2.5µg/ml to check cell viability and analyzed by flow cytometry to measure cell proliferation of the enriched Tcell population through the degree of dilution of CFSE. igM-Binding capacity We assessed the IgM-binding capacity of gill CD8+ DCs. For this, rainbow trout IgM was purified from serum by affinity chromatography, using Econo-Column Chromatography columns (BIO-RAD) and CNBr-activated Sepharose 4b (GE Healthcare), coated with anti-trout IgM mAb (1.14), according to the manufacturer’s instructions. Thereafter, gill leukocytes were incubated with 0.1µg/ml purified IgM for 1h at 20°C. Subsequently, cells were washed twice with L-15 with 5% FCS and then stained with anti-trout CD8α and anti-trout MHC II β-chain as described earlier, in combination with an anti-trout IgM [mAb 1.14 mouse IgG1 coupled to fluorescein (FITC); 1 µg/ml] (30). Cells were washed twice, resuspended in L-15 with 5% FCS, and analyzed on a FACSCalibur flow cytometer. cD40l stimulation The nucleotide sequence corresponding to the extracellular domain of the rainbow trout CD40L (GenBank Accession number EF160131) together with an N-terminal 6× histidine tag was synthetized and subcloned into the E3 expression vector (Abyntek). The recombinant plasmid was transformed into BL21 cells, and kanamycin-resistant single positive colonies for each clone were then incubated at 37°C in Luria–Bertani media. When the OD600 reached 0.6, 0.1mM of isopropyl β-d-thiogalactoside (IPTG, Sigma Aldrich) was added to induce protein production. After 16 h, cells were harvested, lysed by sonication and dissolved using urea. Thereafter, recombinant CD40L was obtained through the use of Nickel columns (Sigma Aldrich). The proteincontaining fractions were pooled, refolded, filtered through 0.22µm, and resuspended in storage buffer (50mM Tris–HCl, 150 mM NaCl, 10% glycerol, and 0.5 M l-arginine, pH 9.0). Protein concentrations were determined in a BCA protein assay (ThermoFisher Scientific), and the recombinant rainbow trout CD40L (85% purity) aliquoted and stored at −80°C until used. An irrelevant protein with a similar molecular weight also bearing an N-terminal His tag was produced in the same conditions and was used as a functional control (C-His). To establish the effect of CD40L on gill CD8+ DCs, gill leukocytes were cultured with 5 µg/ml CD40L, with the same concentration of C-His or with media alone. This dose had been previously optimized in functional assays performed with Bcells in our laboratory (data not shown). After 48h of incubation at 20°C, the percentage of CD8+ DCs and their levels of expression of surface MHC II were analyzed by flow cytometry as described earlier. statistical analysis Statistical analyses were performed using a two-tailed Student’s t test with Welch’s correction when the F test indicated that the variances of both groups differed significantly. The differences between the mean values were considered significant on different degrees, where * means p≤0.05, ** means p≤0.01, and *** means p≤0.005. resUlTs identification of cD8+ Dcs in the gill of rainbow Trout Because in mammals cross-presenting DCs are also found in the lungs, we examined whether a CD8+ DC subset equivalent to that previously reported in rainbow trout skin (10) was also found in gills. For this, gill leukocytes were analyzed by flow cytometry, using anti-trout MHC II and anti-trout CD8α mAbs, and compared with the profile observed in splenocytes. Cells were first analyzed according to their FSC/SSC profile (Figure 1A, left panels), dividing the cells into those that are included within the lymphoid gate (FSClowSSClow) representing lymphocytes, or within what we have cataloged as a myeloid gate (FSChigh SSCmed/high), representing larger and more complex cells, such as macrophages, neutrophils, and DCs (10, 31). Within the myeloid gate, a subset of cells expressing surface CD8 and high levels of surface MHC II cells were found in the gills, representing 1.29% of the cells in the myeloid gate (Figure 1A, top panels), in a similar trend to that seen on the spleen, where this subset was also found in a similar percentage (Figure1A, bottom panels). Some cells expressing CD8+ and lower surface MHC II levels were also observed in both gills and spleen, but in this work we focused exclusively on those expressing high surface MHC II levels such as those previously identified in skin and designated as CD8+ DCs (10). CD8+MHC II+ cells were also found in the lymphoid population of gill leukocytes in high numbers, but this population was almost absent in the spleen. However, the characterization of this lymphoid CD8+MHC II+ subset was not the aim of this study, and it is something we will address in future investigations. Confocal microscopy analysis of gill sections stained with fluorescently labeled antibodies against CD8 and MHC II FigUre 1 | Identification and characterization of gill CD8α+ major histocompatibility complex (MHC) II+ cells. (a) Flow cytometry analysis of rainbow trout leukocytes isolated from gills (top panels) and spleen (bottom panels) stained with anti-CD8α and anti-MHC II mAbs. FSC/SSC profiles are shown (left) and gates for lymphoid (L) and myeloid (M) cells defined. Two-color CD8/MHC class II dot plots of lymphoid and myeloid gated cells are also shown. Percentage of myeloid CD8+MHC IIhi cells among the total number of cells in each gate is shown in the upper right corner. (B) Cryostat sections were prepared from rainbow trout gill, fixed and labeled with anti-CD8α (green) and anti-MHC class II (red) Abs, counterstained with DAPI (blue), and analyzed by fluorescence microscopy. A representative image is shown, together with a magnification of a CD8+ dendritic cell (DC) (right). (c) CD8α+MHC II+ cells from gills were isolated by cell sorting and then incubated onto poly-llysine-treated glass slides, fixed, mounted, and analyzed by light microscopy (scale bar, 5µm). (D) Gill CD8+ DCs and splenic CD8+ Tcells were isolated by flow cytometry and RNA obtained. RNA was also obtained from the RTS11 monocyte–macrophage cell line. These RNAs were used to study the levels of transcription of different marker genes by real-time PCR. Relative expression of the indicated genes to the endogenous control elongation factor 1α was calculated for each sample value. Mean values (+SD) from three independent experiments are shown. Asterisks in red indicate significant differences between values obtained in gill CD8+ DCs and splenic CD8+ Tcells, whereas asterisks in blue denote significant differences between values obtained in gill CD8+ DCs and RTS11 cells (*p≤0.05, **p≤0.01, and ***p≤0.005). 5 Soleto et al. Cross-Presenting DCs in Teleost Gills Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 59 revealed that these large CD8+ cells with high levels of MHC II were primarily located at the distal part of secondary gill filaments (Figure1B; Figure S1 in Supplementary Material). To characterize the morphology of these cells, they were sorted by flow cytometry and analyzed by light microscopy (Figure1C). Cells showed a round irregular morphology with small membrane projections, FigUre 2 | Phagocytic capacity of gills CD8+ dendritic cells (DCs). Leukocytes from gills were incubated with Crimson Red fluorescent polystyrene beads (1µm diameter) at a ratio of 1:10 (cell/beads) for 16h. Non-ingested beads were removed in a glucose gradient, and the cells were stained with anti-CD8α and analyzed by flow cytometry (a). Lymphoid cells (upper panels) and myeloid cells (lower panels) were gated, and CD8− and CD8+ cells were further selected to analyze and measure the fluorescence of internalized beads (histograms). The percentage of cells containing beads was determined using control samples without beads to establish the phagocytosis gate shown in the histograms. The average percentage of phagocytic CD8− and CD8+ cells (B) and the mean fluorescence intensity (MFI) of the internalized beads (c) were used to determine the phagocytic capacity of each cell type. Data are representative of 11 individual fish from 5 different experiments. (D) Cytochalasin (0.05µg/ml) was added to the cells immediately before the addition of the beads to demonstrate active phagocytosis. Results are shown as the percentage of phagocytic gill CD8+ DCs relative to that of untreated controls (five individual fish) (***p≤0.005). 6 Soleto et al. Cross-Presenting DCs in Teleost Gills Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 59 as previously described for mammalian DCs. To further characterize this CD8+ DC population identified in the gills, we analyzed the transcription levels of specific Tcell and DC markers by real-time PCR comparing them to the transcription profiles obtained for CD8+ cytotoxic T cells from spleen (CD8+MHC II− from the lymphoid gate) and the trout macrophage cell line RTS11. We observed that the gill CD8+ DCs expressed DC-SIGN (Figure1D), confirming they constitute a DC subset. In addition, they also expressed very high levels of CD83, CD80/86, and BAFF when compared with CD8+ Tcells or macrophages, also supporting their identification as a DC subpopulation. In concordance, these CD8+ DCs express very low levels of TCRα and CD3 compared with the levels obtained in spleen CD8+ Tcells (approximately 100-fold lower). Interestingly, the transcriptional profile obtained also pointed out some differences between gill CD8+ DCs and the equivalent subset previously identified in trout skin. For example, gill CD8+ DCs did not express CD11b whereas skin CD8+ DCs did; and showed expression levels of CD8β comparable with those found on CD8+ Tcells, indicating that this DC subset was CD8α/β, contrarily to the skin DC subset, which was reported as CD8α/α (10). Phagocytic capacity of Teleost gill cD8+ Dcs Trout gill CD8+ DCs displayed a high phagocytic capacity, in contrast to lymphoid CD8+ cells that were not phagocytic (Figures2A,B). After 16h of incubation with 1µm Crimson red-labeled polystyrene beads, ~60% of the CD8+ DCs had internalized beads, whereas only ~8% of the CD8− fraction within the myeloid gate had internalized beads (Figures2A,B). In addition, the mean fluorescence intensity (MFI) of internalized beads within CD8+ DCs was significantly higher than that observed in CD8− cells within the myeloid gate (Figure2C), indicating that the average number of particles internalized per cell was much higher for CD8+ DCs than for other APCs present in the gills. The internalization of beads by trout gill CD8+ DCs was significantly inhibited by cytochalasin B demonstrating FigUre 3 | Tcell-activating capacity of gill CD8+ dendritic cells (DCs). Trout gill CD8+ DCs were isolated by flow cytometry. Cells were then cultured for 12h in L-15 medium supplemented with 20% fetal calf serum in presence of the Tcell-dependent Ag TNP hapten conjugated to the keyhole limpet hemocyanin. Thereafter, DCs were cocultured with isogenic Tcell-enriched fractions obtained from the spleen of these same animals [auto-mixed leukocyte reaction (MLR)], previously labeled with CFSE. Cocultures were set at a ratio of 1:30 (DC:Tcells) and incubated for 5days. After this time, the cultures were analyzed by flow cytometry to measure the level of CFSE dilution. (a) A representative example from eight individuals is shown, and the percentage of proliferating cells is indicated. (B) Average percentages of proliferating cells were calculated (n=8, mean+SD) (**p≤0.01). 7 Soleto et al. Cross-Presenting DCs in Teleost Gills Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 59 this is an active phagocytosis process that requires actin polymerization (Figure 2D). In addition, the fact that the beads detected in flow cytometry were located always inside the cells was also verified through confocal microscopy (Figure S2 in Supplementary Material). These results clearly point out that trout gill CD8+MHC II+ cells within the myeloid gate are, in fact, phagocytic CD8+ DCs. Teleost gill cD8+ Dcs Prime Proliferation of T cells One of the main defining features of DCs is their capacity to stimulate Tcells (32). Thus, to demonstrate their Tcell-activating potential, we performed an MLR. To carry this out, we FACS isolated gill CD8+MHC II+ cells and incubated them overnight in the presence of TNP-KLH (a T cell-dependent antigen). This CD8+ DC subpopulation was co-incubated with isogeneic (auto-MLR) splenocyte cultures enriched in Tcells from which all APCs had been previously depleted. This enriched population strongly proliferated in the presence of ConA (Figure S1 in Supplementary Material), a specific Tcell mitogen (33), thus confirming the Tcell enrichment but was not able to proliferate in response to TNP-KLH alone (Figure S3 in Supplementary Material). However, when this Tcell-enriched population was cocultured with gill CD8+ DCs, a significant Tcell proliferation was detected by means of CFSE dilution, when compared with Tcell-enriched splenocytes incubated in the absence of gill DCs (Figures3A,B). These data demonstrate a capacity for the teleost gill CD8+ DC subpopulation to activate Tlymphocytes. gill cD8+ Dcs express high levels of surface ccr7 In the mammalian respiratory system, the mobilization of DCs from the lungs to draining LNs is dependent on the chemokine receptor CCR7 and its ligands (34). Because there are no LNs in teleost fish, it has been proposed that secondary immune responses are locally orchestrated in the mucosal tissues that host APCs, B and Tcells dispersed throughout the tissue (35). In this context, we decided to evaluate the levels of CCR7 surface expression in gill CD8+ DCs. In trout gills, no CCR7+ cells were found within the lymphocyte compartment (Figures 4A,B). On the other hand, the percentage CCR7+CD8+ DCs was significantly high (around 50%) compared with the amount of CCR7+ cells in the rest of the myeloid compartment (around 20%) (Figures4A,B). In addition, a significantly higher CCR7 mean fluorescence intensity within the CD8+ DC subpopulation indicates that the density of CCR7 molecules on the cell surface is highest in this gill subset (Figures4A,C). gill cD8+ Dcs express Tlrs and crossPresenting Dc Markers Toll-like receptors are abundantly expressed on DCs since the recognition of molecular signatures from potential pathogens via TLRs is an essential step for DC activation that leads to the initiation of adaptive immunity (36). Hence, we analyzed the levels of transcription of all TLR genes identified in rainbow trout in gill CD8+ DCs, namely, TLR1, TLR2, TLR3, TLR5, TLR7, TLR8a, TLR9, and TLR22. TLR22 is a fish-specific cell surface sensor of dsRNA (37). Our results revealed that gill CD8+ DCs expressed high levels of TLR1, TLR5, TLR7, TLR8a, TLR9, and TLR22, together with intermediate levels of TLR2 and TLR3 (Figure5A). The levels of expression of all TLR genes were much higher in gill CD8+ DCs than in RTS11 macrophages (Figure5A). Concerning splenic CD8+ Tcells, to our surprise, these cells also expressed high levels of most TLRs, with the exception of TLR3 (Figure5A). As TLR3 is considered a signature marker for crosspresenting DCs including murine tissue CD103+ and lymphoid CD8+ DCs, and the human CD141+ DC lineage (16, 20, 21), our results point to the cross-presenting nature of our DC population. To further confirm this point, we examined the levels of transcription of additional exclusive markers associated with human FigUre 4 | Expression of surface CCR7 on gill CD8+ dendritic cells (DCs). Trout leukocytes from gills were isolated, stained with specific Abs against CD8α, major histocompatibility complex (MHC) II and CCR7, and analyzed by multicolor flow cytometry. (a) Cells were gated as lymphoid and myeloid on the basis of their FSC and SSC. Then, CD8+MHC II+ cells were further gated on those populations, and CCR7 fluorescence was determined in CD8+ DCs (open line histograms) and compared against the general CCR7 levels in each gate (filled line histograms). Numbers on plots correspond to the percentage of CCR7+ cells after exclusion of negative cells present on the isotype controls. Average percentage of CCR7 cells (B) and MFI of CCR7 expression (c) in gill lymphoid and myeloid populations were calculated. Data are representative of nine individual fish from three independent experiments (*p≤0.05 and **p≤0.01). 8 Soleto et al. Cross-Presenting DCs in Teleost Gills Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 59 and murine subsets of cross-presenting DCs. We confirmed that gill CD8+ DCs also transcribed CD103, CD141, Baft3, and IRF8 (Figure5B), strongly suggesting that the CD8+ DC population identified in trout gills also corresponds to a cross-presenting DC subset. gill cD8+ Dcs have igM-Binding capacity In mammals, it is known that most innate immune cells express Fc receptors that bind monomeric or aggregated Igs, immune complexes, and opsonized (antibody-coated) particles or cells (38). In DCs, engagement of FcR induces maturation and dramatically increases the efficiency of cross-presentation (39). As IgM is the prevalent Ig in the trout serum and gill mucus (40), we decided to analyze the capacity of the gill CD8+ DC subset to bind serum IgM. Our data revealed that ~31% of CD8+ DCs were able to bind purified IgM, while only ~5% of the rest of the cells within the myeloid gate bound IgM (Figures6B,D). In the case of the cells contained within the lymphoid gate, there were a number of positive cells for IgM staining that should correspond to IgM+ B cells present in the gills (~2%); however, the IgM-binding capacity was negligible in lymphoid CD8+MHC+ cells from this gate (Figures6A,C). gill cD8+ Dcs Produce and are activated by cD40l It has been previously reported that engagement of CD40 by CD40L makes DCs more effective APCs, inducing the upregulation of MHC II and co-stimulatory molecules such as CD80 and CD86 (41). Thus, we aimed to analyze whether fish DCs respond FigUre 5 | Transcription of toll-like receptor (TLR) genes and markers of mammalian cross-presenting dendritic cells (DCs) on gill CD8+ DCs. CD8+ DCs and splenic CD8+ Tcells were isolated by flow cytometry, and RNA obtained. RNA was also obtained from the RTS11 monocyte–macrophage cell line. These RNAs were used to study the levels of transcription of the different TLRs (a) and other markers of cross-presenting DCs (B) by real-time PCR. Relative expression levels of the indicated genes to the endogenous control elongation factor 1α were calculated for each sample value (mean+SD; n=3–7). Asterisks in red indicate significant differences between values obtained in gill CD8+ DCs and splenic CD8+ Tcells, whereas asterisks in blue denote significant differences between values obtained in gill CD8+ DCs and RTS11 cells (*p≤0.05 and **p≤0.01). FigUre 6 | IgM-binding capacity of gill CD8+ dendritic cells (DCs). Gill leukocytes were incubated with purified IgM for 1h at 20°C. Thereafter, cells were stained with specific Abs against CD8α, major histocompatibility complex (MHC) II, and IgM and analyzed by flow cytometry. Leukocytes were gated as lymphoid (a) and myeloid (B) cells on the basis of their FSC and SSC. Then, CD8+MHC II+ cells were further gated on those populations, and the IgM-binding capacity determined in CD8+ DCs (open line histograms) and compared against the general levels of IgM binding in lymphocytes and myeloid cells (filled line histograms). Data represent the IgM+ cells population after exclusion of negative cells present on the isotype controls. (c,D) Average percentage of IgM+ cells (left panels) and the MFI of IgM (right panels) within lymphoid (c) and myeloid (D) cells are shown. Data are representative of seven individual fish from two independent experiments (**p≤0.01). 9 Soleto et al. Cross-Presenting DCs in Teleost Gills Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 59