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Technical Advance: Live-imaging analysis of human dendritic cell migrating behavior under the influence of immune-stimulating reagents in an organotypic model of lung Anh Thu Nguyen Hoang,*,1 Puran Chen,*,1 Sofia Björnfot,* Kari Högstrand,*,2 John G. Lock,† Alf Grandien,*,2 Mark Coles,‡ and Mattias Svensson*,3 *Center for Infectious Medicine, Department of Medicine, and †Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden; and ‡Centre for Immunology and Infection, Hull York Medical School and Department of Biology, University of York, United Kingdom RECEIVED MAY 30, 2013; REVISED APRIL 12, 2014; ACCEPTED MAY 18, 2014. DOI: 10.1189/jlb.3TA0513-303R
ABSTRACT
Introduction
This manuscript describes technical advances allowing manipulation and quantitative analyses of human DC migratory behavior in lung epithelial tissue. DCs are hematopoietic cells essential for the maintenance of tissue homeostasis and the induction of tissue-specific immune responses. Important functions include cytokine production and migration in response to infection for the induction of proper immune responses. To design appropriate strategies to exploit human DC functional properties in lung tissue for the purpose of clinical evaluation, e.g., candidate vaccination and immunotherapy strategies, we have developed a live-imaging assay based on our previously described organotypic model of the human lung. This assay allows provocations and subsequent quantitative investigations of DC functional properties under conditions mimicking morphological and functional features of the in vivo parental tissue. We present protocols to set up and prepare tissue models for 4D (x, y, z, time) fluorescence-imaging analysis that allow spatial and temporal studies of human DCs in live epithelial tissue, followed by flow cytometry analysis of DCs retrieved from digested tissue models. This model system can be useful for elucidating incompletely defined pathways controlling DC functional responses to infection and inflammation in lung epithelial tissue, as well as the efficacy of locally administered candidate interventions. J. Leukoc. Biol. 96: 481– 489; 2014.
DCs are located alongside all epithelial linings of body surfaces [1] and are well-equipped to migrate within and between various tissues and organs [2]. By responding to TLR agonists, DCs determine how immune responses to pathogens are initiated and completed. Additionally, TLR activation contributes to disease pathogenesis in noninfectious pulmonary disorders, including airway disease, acute lung injury, and interstitial lung disease [3]. Within lung, DCs mainly associate with the epithelial layer, and there is evidence of DC regulation by the epithelium and that epithelial dysfunction leads to overzealous immune cell activation [4, 5]. In humans, however, studies on DC immune activation in response to provocation at the tissue site are generally difficult. Therefore, improved human tissue models recapitulating normal tissue that allows analysis of fundamental immunologic traits predisposing to immune activation and host-pathogen interactions, as well as the efficacy of adjuvants and immunomodulatory regimens, are needed. There is evidence demonstrating that adhesion and migration are markedly different in cells within 3D environments compared with monolayer-based cultures [6]. For example, 3D lung-tissue models of normal airway mucosa have proven to be beneficial in studying the behavior of epithelial cells [7–9]. Furthermore, 3D-tissue models have proven to be valuable in examining the importance of cell– cell contact and cell-surface attachment in regulating the proliferation and differentiation of hematopoietic cells in specific tissue niches [10]. These assays can be useful for observing gene expression over time,
Abbreviations: 3D/4D⫽three/four-dimensional, 16HBE⫽human bronchial epithelial cell line 16HBE14o-, APC⫽allophycocyanin, DC⫽dendritic cell, DDAO-SE⫽dodecyldimethylamine oxide-succinimidyl ester, EpCAM⫽ epithelial cellular adhesion molecule, IRES⫽internal ribosome entry site, IVIG⫽intravenous immunoglobulin, Pam3CSK4⫽palmitoyl-3-cysteine-serine-lysine-4, pMSCV⫽plasmid murine stem cell virus, qPCR⫽quantitative polymerase chain reaction The online version of this paper, found at www.jleukbio.org, includes supplemental information.
0741-5400/14/0096-481 © Society for Leukocyte Biology
1. These authors contributed equally to this work. 2. Current address: Center for Hematology and Regenerative Medicine, Dept. of Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden. 3. Correspondence: Center for Infectious Medicine, F59, Dept. of Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden. E-mail: [email protected]
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studying the effects of alterations in stromal cell gene expression, carrying out analyses in response to pathogenic stimuli, as well as carrying out multiple replicates with increased throughput rate. Similarly, establishment and exploitation of 3D-tissue models with immune cells, such as DCs, monocytes, and macrophages, hold great promise in allowing researchers to perform immunologicalbased assays and determining the mechanisms that control hu-
man immune cell function in live tissue [11–13]. Altogether, the multicellular assembly provides secreted factors and multiple cellto-cell communications within the tissue microenvironment that are likely to play an essential role in regulating physiological as well as pathophysiological conditions [14]. We show here that an organotypic model of the human lung (illustrated in Fig. 1A) can be used to study DC activities
Figure 1. Illustration of the lung-tissue model set up and mounting of model for liveimaging experiments. (A) Schematic drawing of our human lung-tissue model composition, showing DC interaction with the stratified lung epithelial layer and fibroblasts in response to microbial stimuli. (B) Schematic drawing of the model set up in a six-well insert from Day 1 to the time of live imaging, totally 14 –16 days. Day 1: culture MRC-5 fibroblasts in bovine collagen type I for 7 days in a six-well insert. Day 7: add monocyte-derived DCs on top of the fibroblast-collagen layer. Day 8: seed 16HBE epithelial cells on top of the DCfibroblast layer, and submerge the culture in medium for 3 days. Day 11: culture the model in an air-liquid interface for 3–5 days. Days 14 –16: stimulate the models for live-imaging experiments. (C–L) The mounting of the model for live imaging. (C) Prewet the glass-bottom well using the apical supernatant from the tissue model. (D) Remove the model from the insert with a sharp scalpel, and place it on a petri dish. (E and F) Transfer the model to the glass-bottom culture plate using a cell lifter and forceps. (G) Place the model with the apical side down toward the glass of the plate, and make sure there are no air bubbles between the model and the glass. (H–J) Add a metallic washer on top of the model, and glue the model to the glass-bottom well by applying tissue adhesive at the outer edge. (K) Add 4% agarose around the washer for additional stabilization. (L) Add 2 ml complete DMEM without phenol red to the mounted model.
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in real time. The model is designed to allow live imaging of DC behavior in lung tissue under next to in vivo settings and is adapted from our previously described physiological 3D-organotypic lung model [12]. This approach is amendable to manipulation of epithelial cell gene expression, as well as the visualization of DC migratory behavior and activation during steady state and under inflammatory conditions.
MATERIALS AND METHODS
Culturing and expansion of cells MRC-5, human fetal lung fibroblasts (American Type Culture Collection, Manassas, VA, USA), frozen at passage 24, were expanded 7 days before setting up the 3D model, as described previously [12]. To generate DCs, human blood monocytes were cultured in CSF-2 and IL-4 for 6 days, as described previously [12]. 16HBE [9], immortalized with the large SV40 T antigen (a gift from Dr. Dieter Gruenert, Mt. Zion Cancer Center, University of California, San Francisco, CA, USA), was thawed and cultured 5–7 days before use in the 3D-tissue models, as described previously [12]. Ethical permission for the use of human cells was obtained from the regional Ethics Committee at the Karolinska Institutet (Stockholm, Sweden).
Expression of fluorescent proteins in 16HBE and MRC-5 cells GFP retroviral particles were produced by transient transfection of 5 ⫻ 106 Phoenix-Ampho packaging cells (a gift from Dr. G. P. Nolan, Stanford University, Stanford, CA, USA), with 10 g pMSCV-IRES-GFP (a gift from Dr. A. Nienhuis, St. Jude Children’s Research Hospital, Memphis, TN, USA) or pMSCV-IRES-orange in the presence of 20 l Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). The pMSCV-IRES-vector and the orange reporter cassette were gifts from Drs. A. Nienhuis and Y. R. Tsien (University of California, Los Angeles, CA, USA), respectively, and the pMSCV-IRES-orange construct was a gift from Dr. Robert Wallin (Karolinska Institutet). Supernatants containing retroviral particles were harvested at 48 h and 72 h after transfection and filtered through a 0.45-m syringe filter. MRC-5 fibroblasts and 16HBE epithelial cells were seeded in six-well plates and cultured until 50% confluent (2–3 days). The MRC-5 fibroblasts were then transduced overnight with 5 ml MSCV-IRES-orange supernatant in the presence of 8 g/ml polybrene (Sigma, St. Louis, MO, USA), and the 16HBE epithelial cells were transduced with 5 ml MSCV-IRES-GFP supernatant. After transduction, the cells were expanded and sorted for positive GFP or Orange expression using BD FACSAria cell sorter with standard filters (BD Biosciences, San Diego, CA, USA).
Setting up 3D lung-tissue models with DCs To set up the organotypic models of the human lung, fibroblasts, DCs, and epithelial cells were added step-by-step following the procedure described previously [12]. Briefly, the amount of reagents needed to establish the fibroblast matrix layer was calculated according to Supplemental Table 1. The reagents were mixed gently in a 50-ml conical polyprophylene tube, ensuring no air-bubble formation, and the tube was kept on ice under sterile conditions at all times. MRC-5 cells were allowed to remodel the collagen for 7 days before DCs were added, and before implantation, DCs were labeled with a cell tracker dye, CellTrace Far Red DDAO-SE (Life Technologies), according to the manufacturer’s instructions. One day after DC implantation, 16HBE cells were added, and the culture medium was changed every 2nd day after epithelial cell addition. The models were kept submerged for 3 days before air exposure. From Day 3 of air exposure, the organotypic models were stimulated and analyzed using live-imaging fluorescence confocal microscopy or harvested for digestion followed by qPCR and flow cytometry analyses.
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Preparation of 3D-tissue models for live imaging To induce inflammatory responses, LPS (TLR4 agonist; InvivoGen, San Diego, CA, USA), Pam3CSK4 (TLR1/2 agonist; InvivoGen), or human rCCL2 (CCR2 agonist; R&D Systems, Minneapolis, MN, USA) was diluted in PBS and applied at the apical side of the tissue model in aliquots of 50 l (Fig. 1B). The final amounts of agonists were 2, 20, 100, 200, or 1000 ng for LPS; 10, 100, or 1000 ng for Pam3CSK4; and 200 ng for CCL2. Then, stimulated or unstimulated (PBS only) tissue models were incubated in 5% CO2 at 37°C for 30 min to allow the stimuli to absorb into the model. After 30 min incubation, the liquid remaining on the apical side of the model was removed gently and used to prewet (prevent air-bubble formation) the six-well, glass-bottom culture plate (MatTek, Ashland, MA, USA), in which the tissue models were mounted (Fig. 1C). By using a sharp scalpel, the models were cut out from the insert and placed on a petri dish (Fig. 1D). Then, the models were transferred to the prewet, glass-bottom culture plate with the apical side facing down toward the glass-bottom well (Fig. 1E–G). A metallic washer was added on top of the model, and the model was glued to the glass by applying tissue adhesive at the outer edge (Fig. 1H–J). Four percent agarose was added around the washer for additional stabilization (Fig. 1K). Complete DMEM (2 ml), without phenol red, was added to the mounted model (Fig. 1L). The models were then incubated for 3 h in 5% CO2 at 37°C for acclimatization and stabilization.
Live imaging and quantification of DC migratory behavior 4D time-lapse images were generated by acquiring 3D z-stacks with a 4-m z resolution in an image volume of 512–948 m in x, y and 120 –140 m in z direction. Image stacks of the tissue models were acquired in all six wells consecutively by scanning each model with an interval of 20 min for each time-point. The time-lapse experiment was performed over 12–16 h with a 20⫻ air objective (Plan Apo VC 20⫻ differential interference contrast N2, numerical aperture 0.80) in 5% CO2 at 37°C using a Nikon A1R spectral detector confocal microscope. Images were acquired using the resonant scanner and the DU4 detector with the 450/50, 525/50, and 595/59 filters. GFP, Orange, and Far Red DDAO-SE was detected with the 488-, 561-, and 647-nm laser lines, respectively. The Imaris software, version 7.6.1 (Bitplane, Zurich, Switzerland), was used for cell tracking, displacement, speed, and sphericity analysis. Only tracks with a displacement above 2.5 m are included in the analysis. For drift correction, cell debris (round, immobile) was used as internal reference and later excluded from the analysis. The distance between DC and the epithelial cell layer in the z direction was quantified using Vantage plot in the Imaris software. For this purpose, individual objects were identified for the epithelial cells using the Surfaces function, and the median z position was calculated in Vantage. Similarly, individual DC objects were generated using Surfaces function. The z differences between each DC and the “average” of the epithelial cells were calculated using Vantage and Excel. Statistical analysis was calculated using GraphPad Prism, applying one-way ANOVA with Kruskal-Wallis test. The analyses were based on at least two separate experiments with monocytederived DCs from at least three different donors in each experiment.
Cytokine measurement Supernatants from tissue model cultures were collected for protein measurement after 16 h of stimulation. To detect TNF, a human TNF DuoSet ELISA kit (R&D Systems) was used, according to the manufacturer’s protocols.
Harvesting 3D models for qPCR and DC flow cytometry analysis To analyze gene expression in tissue models, whole-tissue models were collected and total RNA extracted using the RiboPure kit (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer’s protocol. The geneexpression levels were determined as described previously [12]. The primers and probes for TNF, IL-1, CXCL8, and CCR2 were purchased as Pre-
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Developed TaqMan gene-expression assays (Applied Biosystems). GAPDH (Applied Biosystems) served as an endogenous control to normalize the amount of sample cDNA. To analyze DCs from organotypic models by flow cytometry, the model tissue was cut out from the transwell inserts using a sterile scalpel and put in a small petri dish (5 cm in diameter). Each model was cut into small pieces, ⬃1 mm2/piece, following removal of the membrane. Six ml of 1.5 mg/ml collagenase A (Roche, Switzerland) in complete DMEM was added to the petri dish. Then, the mixture of collagenase and tissue pieces was transferred to a 50-ml conical polyprophylene tube (BD Biosciences), placed in a 37°C water bath with magnetic stirring for 25 min. Next, the dissolved tissue pieces were passed through a 70-m cell strainer (BD Biosciences), placed on top of a 50-ml conical polyprophylene tube to get a single-cell suspension. Then, 7 ml 5 mM EDTA (Sigma) in PBS was passed through the cell strainer into the cell-collagenase suspension. After centrifugation (300 g for 5 min), the supernatant was discarded, and cells were washed once with 5 mM EDTA in PBS. For flow cytometry, cells were resuspended in FACS buffer (PBS with 2% FCS and 1 mM EDTA) and incubated with 1 g/ml IVIG for 10 min on ice, followed by the addition of direct-conjugated antibodies. The following antibodies were used: anti-CD45 BV421 and anti-EpCAM Alexa Flour 647 (BioLegend), anti-HLA-DR PE and anti-CD86 FITC (BD Biosciences), and anti-CCR2 APC (BioLegend). Antibody staining was performed in combination with an APC/Cy7 Live/Dead cell marker (BD Biosciences). Two-hundred thousand cells were stained in 50 l buffer for 30 min at 4°C, followed by washing and fixation with BD Cytofix (BD Biosciences) for 10 min at room temperature. Samples were acquired by using a Fortessa flow cytometer (BD Biosciences), followed by data analysis with FlowJo software (Tree Star, Ashland, OR, USA).
RESULTS AND DISCUSSION
Epithelial cells and fibroblasts expressing fluorescent proteins reveal intricate cellular networks forming the basis for DC distribution and migration in the organotypic model of the human lung To enable exploration of pathways by which microbial stimuli influence human DC migratory behavior in live lung epithelial
tissue, we further developed our recently established organotypic model of the human lung for live-imaging fluorescence microscopy analysis. The tissue model is based on human lung fibroblast and epithelial cell lines, as well as monocyte-derived DCs. Largely, the model displays characteristics of the human lung tissue, including a stratified epithelium associated with production of adherence- and tight-junction proteins, and a basal membrane composed of extracellular matrix proteins in the boundary between the fibroblast and epithelial layers [12]. For live-imaging experiments, the 16HBE and MRC-5 cells were genetically modified to express GFPs and orange fluorescent proteins, respectively, whereas DCs were labeled with a cell tracker dye before implantation. Live-imaging experiments were performed using an inverted confocal microscope that enables sequential analysis of six models at a time, and DCs were imaged at a depth of up to 150 m from the epithelial surface (Fig. 2A). Confocal image analysis revealed a well-defined, stratified layer of epithelial cells, well-separated from the underlying fibroblast matrix layer. Rarely, fibroblasts were observed to cross the boundary between the two layers and intermix with epithelial cells (Fig. 2B). Similarly, the majority of epithelial cells remained localized to one side of the boundary. Although DCs were found on both sides of the boundary, delineating the fibroblast matrix and epithelial layers, a majority of DCs was associated with the epithelial layer (Fig. 2A and B and Supplemental Video 1). Epithelial cells at the basolateral side (closest to the boundary between fibroblasts and epithelial cells) appeared with an elongated phenotype, largely comparable with fibroblasts in the underlying tissue and different to the epithelial cells residing closer to the apical side of the epithelium (Fig. 2C and D and Supplemental Videos 2 and 3). The elongated phenotype may result from the active interaction with the adjacent basal membrane and the sheet
Figure 2. Structural visualization of the live-tissue model. (A) A representative image of the 3D lung-tissue model with 16HBE epithelial cells (green; GFP), DC (blue; Far Red DDAO-SE), and MRC-5 fibroblasts (red; orange fluorescent protein). A rotated image displaying the z dimension of a 150-m-thick vol is shown (see also Supplemental Video 1). (B) A side-view image of the tissue model displaying a stratified layer of green fluorescent epithelial cells on top of a red fluorescent fibroblast matrix. (C) The boundary between the epithelial layer and the fibroblast matrix (see also Supplemental Videos 2 and 3). (D) Top view fluorescent image of the tissue model showing a homogenous and dense layer of epithelial cells.
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of fibers in which the epithelial cells find their anchoring platform essential to organ function [15]. Together, these data indicate that the organotypic model of the human lung is well-suited for studying the network of processes regulating DC distribution, activation, and migration in a milieu known to regulate DCs, as well as the induction of local immune responses [16].
Stimulation of the organotypic model of the human lung induces relocalization and altered migratory behavior of DCs DCs play an important role in the host by participating in immune surveillance by the expression of TLRs, which determine the magnitude and outcome of immune responses. Although TLR agonists can induce DC maturation directly, other cells in the tissue, such as epithelial cells and fibroblasts, also express TLRs and release cytokines and chemokines that can contribute to induction of DC activation and migration [17]. To verify whether the organotypic model is suitable to monitor DC migration upon stimulation with TLR agonists and a chemokine known to induce DC migration [18], models were stimulated with LPS, Pam3CSK4, or human rCCL2. As shown in Fig. 3A and B, DCs, in models stimulated with Pam3CSK4, distributed less frequently in the fibroblast matrix layer but rather, closer to the center of the epithelial layer when compared with the unstimulated tissue model. The comparison of the distribution of each DC with the epithelial cell layer, 4 h poststimulation, revealed that Pam3CSK4 and CCL2, but not LPS, induced relocation of DCs (Fig. 3C). The tissue models were stimulated with two additional concentrations of LPS (100 ng and 1000 ng), but similarly to 200 ng LPS, neither 100 nor 1000 ng LPS induced detectable relocation of DCs, and the 1000-ng stimulation had
substantial cytotoxic effects, in particular, on the epithelial cells (data not shown). Although no LPS-mediated effect on DC relocalization was observed under these conditions, track-displacement graphs indicated that DCs in LPS-stimulated tissue models explored a wider territory in the x and y plane (data not shown), and analyses of DC displacement revealed that 200 ng was superior to 100 ng in inducing DC motility (Fig. 3D). Thus, the demonstration that DCs in the lung-tissue model respond to LPS-induced inflammation is in line with previous studies that demonstrate a role of the airway epithelium in LPS-induced DC migration in lung tissue [17]. Together, these results may also suggest that distinct TLR agonists influence different pathways involved in directing DC migration at the local site of inflammation and that this may be a result of time-dependent mechanisms and/or whether it is a direct or indirect effect by the TLR agonist on DCs. In addition, the migration of DCs toward the chemotactic CCL2 gradient implies that this approach can be used to validate DC chemokine responses at the local tissue site. This is also in line with our previously published study exploring human monocyte redistribution in the lung-tissue model exposed to CCL2, as revealed by fluorescence microscopy analysis of cryosections from the tissue model with fluorescently labeled monocytes [13]. The modeling of activities, resulting from different provocations in the tissue, may be particularly important in understanding mechanistic actions of adjuvants and other immunestimulatory reagents. It may also serve as a useful tool, demonstrating effects of noxious compounds in underlying clinical manifestation of lung inflammation, as well as testing reagents aimed at blocking myeloid cell activation and migration locally.
Figure 3. Relocation of DCs toward the center of the epithelial cell layer upon stimulation. (A and B) Side-view immunofluorescence images of 140 m z projections of the 3D lung-tissue models with 16HBE epithelial cells (green) and DCs (blue), in the absence (A) or presence (B) of Pam3CSK4 (1000 ng, 4 h). (C) DC migration as distance (z direction) from the center of the epithelial cell layer, 4 h poststimulation with LPS (200 ng), Pam3CSK4 (1000 ng), and CCL2 (200 ng). The analyses were based on three separate experiments. (D) Comparing the extent of DC displacement in response to 100 ng (left) and 200 ng (right) of LPS, based on the parameter track length. Each dot in the graph represents one individual DC, and data are shown with black bars as mean values from one representative experiment ⫾ sem. The statistical significance (one-way ANOVA applying Kruskal-Wallis test) of the indicated P values was determined: *P ⫽ 0.05; ***P ⬍ 0.005; ns ⫽ nonsignificant.
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To elucidate further the influence that inflammatory provocation has on DC cell motility in the lung-tissue models stimulated with LPS, Pam3CSK4, and CCL2, DC migratory behavior was monitored for 16 h. Automated tracking was used to quantitate the displacement for each individual DC (Fig. 4A and B), and the length and speed with which DCs moved under the different conditions applied were quantified. This revealed that DCs moved longer distances in combination with an increase in the mean of average cell
velocities in response to LPS, Pam3CSK4, and CCL2 (Fig. 4C and D). In addition, DCs in stimulated models exhibited a less-spherical shape (Fig. 4E) compared with unstimulated models. The morphological alteration of DCs was most predominant in models stimulated with LPS (Fig. 4E). Furthermore, a reduced spherical morphology correlated with an increase in DC mean velocity in the organotypic lung tissue model (Fig. 4F–I) and is consistent with studies showing that a prerequisite for cellular migration is that cells can
Figure 4. Quantitative analysis of DC migrating behavior in the inflammatory lung microenvironment. (A) A 3D-rendered image of a tissue model showing individual DCs (blue) only. (B) Automated colorcoded tracks of the DCs represent increasing time from start of imaging (blue) to end of imaging (red). Grid-spacing (distance between major tick marks) is 50 m for x, y, and z orientation in all images. (C–E) Comparison of the extent of DC motility with different immune-stimulating reagents, based on the parameter track length, track speed, and track sphericity. The less spherical the cells, the more their spherical measure deviates from the value 1. (F–I) Correlation graphs of track speed mean versus track sphericity mean. All results are representatives of at least three independent experiments. The statistical significance (one-way ANOVA applying Kruskal-Wallis test and correlation analysis in GraphPad Prism, v. 5) of the indicated P values was determined: *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.005, ns ⫽ nonsignificant.
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adopt an elongated morphology [19]. These data, we believe, provide new possibilities in investigating mechanisms of tissue communication circuits of the human lung tissue that underly DC motility in the microenvironment [20].
LPS stimulation of the organotypic model of the human lung induces inflammation and DC activation To verify that TLR agonist stimulations induced an inflammatory environment in the lung-tissue models, we examined the production of TNF in response to LPS and Pam3CSK at the endpoint for the live-imaging analysis (16 h poststimulation). This revealed that LPS, but not Pam3CSK4, induced detectable levels of TNF (Fig. 5A). At this time-point, no increase in TNF mRNA expression was detected in models stimulated with LPS (Fig. 5B) or Pam3CSK4 (data not shown), which is consistent with the fact that TNF is an early transient inflammatory marker, whose transcription level peaks within 1–2 h of stimulation [21]. In contrast, the CXCL8 and IL-1 mRNA tran-
scription levels were elevated in response to LPS at 16 h poststimulation (Fig. 5B), confirming that inflammation persists and may continue to affect the DC in the tissue model. However, we could not detect any difference in the CXCL8 or IL-1 mRNA levels in response to Pam3CSK4 stimulation, despite the fact that we detected enhanced DC mobility in Pam3CSK4-stimulated models (Figs. 3 and 4). The reasons for this can be time-dependent factors, but also, specific regulatory mechanisms might be involved [22], and this needs to be investigated further. To confirm that the course of inflammation induced by LPS was associated with cellular DC activation, DCs were retrieved from tissue models by enzymatic digestion, and individual cells were analyzed for the expression of relevant cell-surface markers by flow cytometry. DCs were identified as CD45⫹ cells with a relatively high surface expression of HLA-DR and a relatively low surface expression of CCR2 (Fig. 5C). At 16 h of LPS stimulation, DCs showed an increased level of surface HLA-DR and
Figure 5. Induction of inflammation and DC activation in the lung-tissue microenvironment stimulated with LPS. (A) The amount of detectable TNF in culture supernatants from tissue models stimulated with LPS or Pam3CSK4 is shown. (B) qPCR analyses of TNF, CXCL8, and IL-1 mRNA expression in unstimulated or LPS-stimulated tissue models. (C) Dot plots (dead cells excluded) and histograms (live CD45⫹ cells) from flow cytometry analyses of cells retrieved from digested tissue models and stained with a Live/Dead cell marker, in combination with anti-CD45, EpCAM HLA-DR, and CCR2 antibodies. SSC, Side-scatter; FSC, forward-scatter. (D and E) The fluorescence intensity or relative mean fluorescence intensity (MFI) of surface HLA-DR (D) and CD86 (E) on DCs (live CD45⫹ cells) retrieved from unstimulated (green) tissue models or tissue models stimulated with various concentrations of LPS, 2 ng (blue), 20 ng (black), and 200 ng (red). (D and E) The histograms represent results from one donor, whereas the bar graphs represents pooled results from six different donors and two independent experiments. The relative expression levels were calculated by subtracting the background mean fluorescence intensity (fluorescence minus one) for each sample and then dividing by the mean fluorescence intensity of the unstimulated sample for the same donor. All results are representatives of at least two independent experiments, and nontransduced 16HBE and MRC-5 ells were used for these experiments. The statistical significance (Mann-Whitney U-test in GraphPad Prism, v.5) of the indicated P values was determined: *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.005.
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CD86, consistent with cellular activation and maturation (Fig. 5D and E). Although these data demonstrated that LPS induces inflammation and DC maturation in the tissue-model system, we cannot conclude, at this stage, whether the LPS acts directly or indirectly on the DC. Nevertheless, these results demonstrate that the tissue-model system can serve as a basis for detailed investigations regarding the prerequisites for DC activation and migration in human lung epithelial tissue. In addition, this organotypic model system provides a robust and reproducible set-up appropriate to identify distinct signaling pathways in human lung tissue relevant to DC function, even beyond cellular migration and activation, in response to TLR agonists and chemokines. Robust and reliable tissue-model systems are key to advance further the field of basic human immunology, as well as the translational exploitation of such basic knowledge. Assays of human DC behavior in live tissue are generally difficult to perform, but this may be overcome by the set-up and use of tissue-model systems, as described here, which resemble the morphological and functional features of their in vivo parental tissues. Model systems should also allow intervention in combination with 4D (x, y, z, time) fluorescence imaging. This is also in line with previous studies that have shown that DC migration is largely influenced by tissue origin, degree of maturity, and the 3D structure of the microenvironment [23]. Although it is becoming evident that tissue homeostasis in the lung is dependent on epithelial cell-mediated regulation of DC [5], the precise mechanisms by which different human DC subsets are regulated during homeostasis and in response to microbial stimuli require more detailed investigations. Therefore, it is important to develop further the existing organotypic model of the human lung to allow the implantation and/or differentiation of distinct DC subsets. Potentially, strategies that involve differentiation of CD34⫹ progenitors under conditions supporting development of precursor DCs [24], which have the potential to generate distinct DC subsets, could be used for implantation into the tissue models. Comprehensive flow cytometry analyses, combined with live-imaging experiments of model tissues stimulated with compounds of distinct material and compositions, can then be applied for in-depth investigations regarding human DC differentiation, activation, and function in tissue. Thus, this model system provides a tool to identify how site-specific stimulation with particular compounds aimed at activating DCs, e.g., adjuvant and vaccine candidates and noxious compounds [25], can be exploited to provoke site-specific DC differentiation, migration, and activation. The quantification of DC redistribution and characterization of DC activation in response to inflammation, we anticipate, can also provide a powerful tool to explore, in detail, the mechanisms dictating DC anchoring and migrating behavior regulated by chemokine receptors and their ligands, present within the lung epithelial cell layer. Furthermore, this model system is also anticipated to provide a means for identifying the efficacy of anti-inflammatory reagents on DC activation during inflammation. Overall, these data demonstrate that this protocol is useful for visualizing processes of human DC migration and activation in models mimicking real tissue and that it is amendable to use with multiple stimuli. 488 Journal of Leukocyte Biology
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AUTHORSHIP A.T.N.H., P.C. and S.B. conducted the experiments. K.H. and A.G. transduced the fibroblast and epithelial cell lines with fluorescent proteins. J.G.L. and M.C. provided supervision on live-imaging data acquisition and analyses. M.S. supervised the study and contributed to the analysis and interpretation of the results. A.T.N.H. and M.S. wrote most parts of the manuscript. All authors contributed to the study design and participated in editing and finalizing the manuscript, which was approved by all authors.
ACKNOWLEDGMENTS Work in the authors’ laboratory is supported by grants from the Swedish Research Council, the Karolinska Institutet, The Knut and Alice Wallenberg Foundation, Stockholm County Council, and The Swedish Fund for Research Without Animal Experiments. P.C. and A.T.N.H. are both recipients of a Karolinska Postgraduate Studentship. This study was, in part, performed at the Live Cell Imaging Unit, Department of Biosciences and Nutrition, Karolinska Institutet (Huddinge, Sweden), supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, and the Centre for Bioscences. The authors thank Salah Zangenah for taking photos illustrating the laboratory work, as well as Aenni Ali and Sylvie Le Guyader for excellent technical assistance with cell cultures and Imaris analysis, respectively.
DISCLOSURES
No conflict of interest, financial or otherwise, is declared by the authors.
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KEY WORDS: 3D-tissue models 䡠 epithelial tissue 䡠 Toll-like receptor agonists 䡠 inflammation
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Technical Advance: Live-imaging analysis of human dendritic cell migrating behavior under the influence of immune-stimulating reagents in an organotypic model of lung Anh Thu Nguyen Hoang,*,1 Puran Chen,*,1 Sofia Björnfot,* Kari Högstrand,*,2 John G. Lock,† Alf Grandien,*,2 Mark Coles,‡ and Mattias Svensson*,3 *Center for Infectious Medicine, Department of Medicine, and †Center for Biosciences, Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden; and ‡Centre for Immunology and Infection, Hull York Medical School and Department of Biology, University of York, United Kingdom RECEIVED MAY 30, 2013; REVISED APRIL 12, 2014; ACCEPTED MAY 18, 2014. DOI: 10.1189/jlb.3TA0513-303R
ABSTRACT
Introduction
This manuscript describes technical advances allowing manipulation and quantitative analyses of human DC migratory behavior in lung epithelial tissue. DCs are hematopoietic cells essential for the maintenance of tissue homeostasis and the induction of tissue-specific immune responses. Important functions include cytokine production and migration in response to infection for the induction of proper immune responses. To design appropriate strategies to exploit human DC functional properties in lung tissue for the purpose of clinical evaluation, e.g., candidate vaccination and immunotherapy strategies, we have developed a live-imaging assay based on our previously described organotypic model of the human lung. This assay allows provocations and subsequent quantitative investigations of DC functional properties under conditions mimicking morphological and functional features of the in vivo parental tissue. We present protocols to set up and prepare tissue models for 4D (x, y, z, time) fluorescence-imaging analysis that allow spatial and temporal studies of human DCs in live epithelial tissue, followed by flow cytometry analysis of DCs retrieved from digested tissue models. This model system can be useful for elucidating incompletely defined pathways controlling DC functional responses to infection and inflammation in lung epithelial tissue, as well as the efficacy of locally administered candidate interventions. J. Leukoc. Biol. 96: 481– 489; 2014.
DCs are located alongside all epithelial linings of body surfaces [1] and are well-equipped to migrate within and between various tissues and organs [2]. By responding to TLR agonists, DCs determine how immune responses to pathogens are initiated and completed. Additionally, TLR activation contributes to disease pathogenesis in noninfectious pulmonary disorders, including airway disease, acute lung injury, and interstitial lung disease [3]. Within lung, DCs mainly associate with the epithelial layer, and there is evidence of DC regulation by the epithelium and that epithelial dysfunction leads to overzealous immune cell activation [4, 5]. In humans, however, studies on DC immune activation in response to provocation at the tissue site are generally difficult. Therefore, improved human tissue models recapitulating normal tissue that allows analysis of fundamental immunologic traits predisposing to immune activation and host-pathogen interactions, as well as the efficacy of adjuvants and immunomodulatory regimens, are needed. There is evidence demonstrating that adhesion and migration are markedly different in cells within 3D environments compared with monolayer-based cultures [6]. For example, 3D lung-tissue models of normal airway mucosa have proven to be beneficial in studying the behavior of epithelial cells [7–9]. Furthermore, 3D-tissue models have proven to be valuable in examining the importance of cell– cell contact and cell-surface attachment in regulating the proliferation and differentiation of hematopoietic cells in specific tissue niches [10]. These assays can be useful for observing gene expression over time,
Abbreviations: 3D/4D⫽three/four-dimensional, 16HBE⫽human bronchial epithelial cell line 16HBE14o-, APC⫽allophycocyanin, DC⫽dendritic cell, DDAO-SE⫽dodecyldimethylamine oxide-succinimidyl ester, EpCAM⫽ epithelial cellular adhesion molecule, IRES⫽internal ribosome entry site, IVIG⫽intravenous immunoglobulin, Pam3CSK4⫽palmitoyl-3-cysteine-serine-lysine-4, pMSCV⫽plasmid murine stem cell virus, qPCR⫽quantitative polymerase chain reaction The online version of this paper, found at www.jleukbio.org, includes supplemental information.
0741-5400/14/0096-481 © Society for Leukocyte Biology
1. These authors contributed equally to this work. 2. Current address: Center for Hematology and Regenerative Medicine, Dept. of Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden. 3. Correspondence: Center for Infectious Medicine, F59, Dept. of Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden. E-mail: [email protected]
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studying the effects of alterations in stromal cell gene expression, carrying out analyses in response to pathogenic stimuli, as well as carrying out multiple replicates with increased throughput rate. Similarly, establishment and exploitation of 3D-tissue models with immune cells, such as DCs, monocytes, and macrophages, hold great promise in allowing researchers to perform immunologicalbased assays and determining the mechanisms that control hu-
man immune cell function in live tissue [11–13]. Altogether, the multicellular assembly provides secreted factors and multiple cellto-cell communications within the tissue microenvironment that are likely to play an essential role in regulating physiological as well as pathophysiological conditions [14]. We show here that an organotypic model of the human lung (illustrated in Fig. 1A) can be used to study DC activities
Figure 1. Illustration of the lung-tissue model set up and mounting of model for liveimaging experiments. (A) Schematic drawing of our human lung-tissue model composition, showing DC interaction with the stratified lung epithelial layer and fibroblasts in response to microbial stimuli. (B) Schematic drawing of the model set up in a six-well insert from Day 1 to the time of live imaging, totally 14 –16 days. Day 1: culture MRC-5 fibroblasts in bovine collagen type I for 7 days in a six-well insert. Day 7: add monocyte-derived DCs on top of the fibroblast-collagen layer. Day 8: seed 16HBE epithelial cells on top of the DCfibroblast layer, and submerge the culture in medium for 3 days. Day 11: culture the model in an air-liquid interface for 3–5 days. Days 14 –16: stimulate the models for live-imaging experiments. (C–L) The mounting of the model for live imaging. (C) Prewet the glass-bottom well using the apical supernatant from the tissue model. (D) Remove the model from the insert with a sharp scalpel, and place it on a petri dish. (E and F) Transfer the model to the glass-bottom culture plate using a cell lifter and forceps. (G) Place the model with the apical side down toward the glass of the plate, and make sure there are no air bubbles between the model and the glass. (H–J) Add a metallic washer on top of the model, and glue the model to the glass-bottom well by applying tissue adhesive at the outer edge. (K) Add 4% agarose around the washer for additional stabilization. (L) Add 2 ml complete DMEM without phenol red to the mounted model.
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in real time. The model is designed to allow live imaging of DC behavior in lung tissue under next to in vivo settings and is adapted from our previously described physiological 3D-organotypic lung model [12]. This approach is amendable to manipulation of epithelial cell gene expression, as well as the visualization of DC migratory behavior and activation during steady state and under inflammatory conditions.
MATERIALS AND METHODS
Culturing and expansion of cells MRC-5, human fetal lung fibroblasts (American Type Culture Collection, Manassas, VA, USA), frozen at passage 24, were expanded 7 days before setting up the 3D model, as described previously [12]. To generate DCs, human blood monocytes were cultured in CSF-2 and IL-4 for 6 days, as described previously [12]. 16HBE [9], immortalized with the large SV40 T antigen (a gift from Dr. Dieter Gruenert, Mt. Zion Cancer Center, University of California, San Francisco, CA, USA), was thawed and cultured 5–7 days before use in the 3D-tissue models, as described previously [12]. Ethical permission for the use of human cells was obtained from the regional Ethics Committee at the Karolinska Institutet (Stockholm, Sweden).
Expression of fluorescent proteins in 16HBE and MRC-5 cells GFP retroviral particles were produced by transient transfection of 5 ⫻ 106 Phoenix-Ampho packaging cells (a gift from Dr. G. P. Nolan, Stanford University, Stanford, CA, USA), with 10 g pMSCV-IRES-GFP (a gift from Dr. A. Nienhuis, St. Jude Children’s Research Hospital, Memphis, TN, USA) or pMSCV-IRES-orange in the presence of 20 l Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). The pMSCV-IRES-vector and the orange reporter cassette were gifts from Drs. A. Nienhuis and Y. R. Tsien (University of California, Los Angeles, CA, USA), respectively, and the pMSCV-IRES-orange construct was a gift from Dr. Robert Wallin (Karolinska Institutet). Supernatants containing retroviral particles were harvested at 48 h and 72 h after transfection and filtered through a 0.45-m syringe filter. MRC-5 fibroblasts and 16HBE epithelial cells were seeded in six-well plates and cultured until 50% confluent (2–3 days). The MRC-5 fibroblasts were then transduced overnight with 5 ml MSCV-IRES-orange supernatant in the presence of 8 g/ml polybrene (Sigma, St. Louis, MO, USA), and the 16HBE epithelial cells were transduced with 5 ml MSCV-IRES-GFP supernatant. After transduction, the cells were expanded and sorted for positive GFP or Orange expression using BD FACSAria cell sorter with standard filters (BD Biosciences, San Diego, CA, USA).
Setting up 3D lung-tissue models with DCs To set up the organotypic models of the human lung, fibroblasts, DCs, and epithelial cells were added step-by-step following the procedure described previously [12]. Briefly, the amount of reagents needed to establish the fibroblast matrix layer was calculated according to Supplemental Table 1. The reagents were mixed gently in a 50-ml conical polyprophylene tube, ensuring no air-bubble formation, and the tube was kept on ice under sterile conditions at all times. MRC-5 cells were allowed to remodel the collagen for 7 days before DCs were added, and before implantation, DCs were labeled with a cell tracker dye, CellTrace Far Red DDAO-SE (Life Technologies), according to the manufacturer’s instructions. One day after DC implantation, 16HBE cells were added, and the culture medium was changed every 2nd day after epithelial cell addition. The models were kept submerged for 3 days before air exposure. From Day 3 of air exposure, the organotypic models were stimulated and analyzed using live-imaging fluorescence confocal microscopy or harvested for digestion followed by qPCR and flow cytometry analyses.
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Preparation of 3D-tissue models for live imaging To induce inflammatory responses, LPS (TLR4 agonist; InvivoGen, San Diego, CA, USA), Pam3CSK4 (TLR1/2 agonist; InvivoGen), or human rCCL2 (CCR2 agonist; R&D Systems, Minneapolis, MN, USA) was diluted in PBS and applied at the apical side of the tissue model in aliquots of 50 l (Fig. 1B). The final amounts of agonists were 2, 20, 100, 200, or 1000 ng for LPS; 10, 100, or 1000 ng for Pam3CSK4; and 200 ng for CCL2. Then, stimulated or unstimulated (PBS only) tissue models were incubated in 5% CO2 at 37°C for 30 min to allow the stimuli to absorb into the model. After 30 min incubation, the liquid remaining on the apical side of the model was removed gently and used to prewet (prevent air-bubble formation) the six-well, glass-bottom culture plate (MatTek, Ashland, MA, USA), in which the tissue models were mounted (Fig. 1C). By using a sharp scalpel, the models were cut out from the insert and placed on a petri dish (Fig. 1D). Then, the models were transferred to the prewet, glass-bottom culture plate with the apical side facing down toward the glass-bottom well (Fig. 1E–G). A metallic washer was added on top of the model, and the model was glued to the glass by applying tissue adhesive at the outer edge (Fig. 1H–J). Four percent agarose was added around the washer for additional stabilization (Fig. 1K). Complete DMEM (2 ml), without phenol red, was added to the mounted model (Fig. 1L). The models were then incubated for 3 h in 5% CO2 at 37°C for acclimatization and stabilization.
Live imaging and quantification of DC migratory behavior 4D time-lapse images were generated by acquiring 3D z-stacks with a 4-m z resolution in an image volume of 512–948 m in x, y and 120 –140 m in z direction. Image stacks of the tissue models were acquired in all six wells consecutively by scanning each model with an interval of 20 min for each time-point. The time-lapse experiment was performed over 12–16 h with a 20⫻ air objective (Plan Apo VC 20⫻ differential interference contrast N2, numerical aperture 0.80) in 5% CO2 at 37°C using a Nikon A1R spectral detector confocal microscope. Images were acquired using the resonant scanner and the DU4 detector with the 450/50, 525/50, and 595/59 filters. GFP, Orange, and Far Red DDAO-SE was detected with the 488-, 561-, and 647-nm laser lines, respectively. The Imaris software, version 7.6.1 (Bitplane, Zurich, Switzerland), was used for cell tracking, displacement, speed, and sphericity analysis. Only tracks with a displacement above 2.5 m are included in the analysis. For drift correction, cell debris (round, immobile) was used as internal reference and later excluded from the analysis. The distance between DC and the epithelial cell layer in the z direction was quantified using Vantage plot in the Imaris software. For this purpose, individual objects were identified for the epithelial cells using the Surfaces function, and the median z position was calculated in Vantage. Similarly, individual DC objects were generated using Surfaces function. The z differences between each DC and the “average” of the epithelial cells were calculated using Vantage and Excel. Statistical analysis was calculated using GraphPad Prism, applying one-way ANOVA with Kruskal-Wallis test. The analyses were based on at least two separate experiments with monocytederived DCs from at least three different donors in each experiment.
Cytokine measurement Supernatants from tissue model cultures were collected for protein measurement after 16 h of stimulation. To detect TNF, a human TNF DuoSet ELISA kit (R&D Systems) was used, according to the manufacturer’s protocols.
Harvesting 3D models for qPCR and DC flow cytometry analysis To analyze gene expression in tissue models, whole-tissue models were collected and total RNA extracted using the RiboPure kit (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer’s protocol. The geneexpression levels were determined as described previously [12]. The primers and probes for TNF, IL-1, CXCL8, and CCR2 were purchased as Pre-
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Developed TaqMan gene-expression assays (Applied Biosystems). GAPDH (Applied Biosystems) served as an endogenous control to normalize the amount of sample cDNA. To analyze DCs from organotypic models by flow cytometry, the model tissue was cut out from the transwell inserts using a sterile scalpel and put in a small petri dish (5 cm in diameter). Each model was cut into small pieces, ⬃1 mm2/piece, following removal of the membrane. Six ml of 1.5 mg/ml collagenase A (Roche, Switzerland) in complete DMEM was added to the petri dish. Then, the mixture of collagenase and tissue pieces was transferred to a 50-ml conical polyprophylene tube (BD Biosciences), placed in a 37°C water bath with magnetic stirring for 25 min. Next, the dissolved tissue pieces were passed through a 70-m cell strainer (BD Biosciences), placed on top of a 50-ml conical polyprophylene tube to get a single-cell suspension. Then, 7 ml 5 mM EDTA (Sigma) in PBS was passed through the cell strainer into the cell-collagenase suspension. After centrifugation (300 g for 5 min), the supernatant was discarded, and cells were washed once with 5 mM EDTA in PBS. For flow cytometry, cells were resuspended in FACS buffer (PBS with 2% FCS and 1 mM EDTA) and incubated with 1 g/ml IVIG for 10 min on ice, followed by the addition of direct-conjugated antibodies. The following antibodies were used: anti-CD45 BV421 and anti-EpCAM Alexa Flour 647 (BioLegend), anti-HLA-DR PE and anti-CD86 FITC (BD Biosciences), and anti-CCR2 APC (BioLegend). Antibody staining was performed in combination with an APC/Cy7 Live/Dead cell marker (BD Biosciences). Two-hundred thousand cells were stained in 50 l buffer for 30 min at 4°C, followed by washing and fixation with BD Cytofix (BD Biosciences) for 10 min at room temperature. Samples were acquired by using a Fortessa flow cytometer (BD Biosciences), followed by data analysis with FlowJo software (Tree Star, Ashland, OR, USA).
RESULTS AND DISCUSSION
Epithelial cells and fibroblasts expressing fluorescent proteins reveal intricate cellular networks forming the basis for DC distribution and migration in the organotypic model of the human lung To enable exploration of pathways by which microbial stimuli influence human DC migratory behavior in live lung epithelial
tissue, we further developed our recently established organotypic model of the human lung for live-imaging fluorescence microscopy analysis. The tissue model is based on human lung fibroblast and epithelial cell lines, as well as monocyte-derived DCs. Largely, the model displays characteristics of the human lung tissue, including a stratified epithelium associated with production of adherence- and tight-junction proteins, and a basal membrane composed of extracellular matrix proteins in the boundary between the fibroblast and epithelial layers [12]. For live-imaging experiments, the 16HBE and MRC-5 cells were genetically modified to express GFPs and orange fluorescent proteins, respectively, whereas DCs were labeled with a cell tracker dye before implantation. Live-imaging experiments were performed using an inverted confocal microscope that enables sequential analysis of six models at a time, and DCs were imaged at a depth of up to 150 m from the epithelial surface (Fig. 2A). Confocal image analysis revealed a well-defined, stratified layer of epithelial cells, well-separated from the underlying fibroblast matrix layer. Rarely, fibroblasts were observed to cross the boundary between the two layers and intermix with epithelial cells (Fig. 2B). Similarly, the majority of epithelial cells remained localized to one side of the boundary. Although DCs were found on both sides of the boundary, delineating the fibroblast matrix and epithelial layers, a majority of DCs was associated with the epithelial layer (Fig. 2A and B and Supplemental Video 1). Epithelial cells at the basolateral side (closest to the boundary between fibroblasts and epithelial cells) appeared with an elongated phenotype, largely comparable with fibroblasts in the underlying tissue and different to the epithelial cells residing closer to the apical side of the epithelium (Fig. 2C and D and Supplemental Videos 2 and 3). The elongated phenotype may result from the active interaction with the adjacent basal membrane and the sheet
Figure 2. Structural visualization of the live-tissue model. (A) A representative image of the 3D lung-tissue model with 16HBE epithelial cells (green; GFP), DC (blue; Far Red DDAO-SE), and MRC-5 fibroblasts (red; orange fluorescent protein). A rotated image displaying the z dimension of a 150-m-thick vol is shown (see also Supplemental Video 1). (B) A side-view image of the tissue model displaying a stratified layer of green fluorescent epithelial cells on top of a red fluorescent fibroblast matrix. (C) The boundary between the epithelial layer and the fibroblast matrix (see also Supplemental Videos 2 and 3). (D) Top view fluorescent image of the tissue model showing a homogenous and dense layer of epithelial cells.
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of fibers in which the epithelial cells find their anchoring platform essential to organ function [15]. Together, these data indicate that the organotypic model of the human lung is well-suited for studying the network of processes regulating DC distribution, activation, and migration in a milieu known to regulate DCs, as well as the induction of local immune responses [16].
Stimulation of the organotypic model of the human lung induces relocalization and altered migratory behavior of DCs DCs play an important role in the host by participating in immune surveillance by the expression of TLRs, which determine the magnitude and outcome of immune responses. Although TLR agonists can induce DC maturation directly, other cells in the tissue, such as epithelial cells and fibroblasts, also express TLRs and release cytokines and chemokines that can contribute to induction of DC activation and migration [17]. To verify whether the organotypic model is suitable to monitor DC migration upon stimulation with TLR agonists and a chemokine known to induce DC migration [18], models were stimulated with LPS, Pam3CSK4, or human rCCL2. As shown in Fig. 3A and B, DCs, in models stimulated with Pam3CSK4, distributed less frequently in the fibroblast matrix layer but rather, closer to the center of the epithelial layer when compared with the unstimulated tissue model. The comparison of the distribution of each DC with the epithelial cell layer, 4 h poststimulation, revealed that Pam3CSK4 and CCL2, but not LPS, induced relocation of DCs (Fig. 3C). The tissue models were stimulated with two additional concentrations of LPS (100 ng and 1000 ng), but similarly to 200 ng LPS, neither 100 nor 1000 ng LPS induced detectable relocation of DCs, and the 1000-ng stimulation had
substantial cytotoxic effects, in particular, on the epithelial cells (data not shown). Although no LPS-mediated effect on DC relocalization was observed under these conditions, track-displacement graphs indicated that DCs in LPS-stimulated tissue models explored a wider territory in the x and y plane (data not shown), and analyses of DC displacement revealed that 200 ng was superior to 100 ng in inducing DC motility (Fig. 3D). Thus, the demonstration that DCs in the lung-tissue model respond to LPS-induced inflammation is in line with previous studies that demonstrate a role of the airway epithelium in LPS-induced DC migration in lung tissue [17]. Together, these results may also suggest that distinct TLR agonists influence different pathways involved in directing DC migration at the local site of inflammation and that this may be a result of time-dependent mechanisms and/or whether it is a direct or indirect effect by the TLR agonist on DCs. In addition, the migration of DCs toward the chemotactic CCL2 gradient implies that this approach can be used to validate DC chemokine responses at the local tissue site. This is also in line with our previously published study exploring human monocyte redistribution in the lung-tissue model exposed to CCL2, as revealed by fluorescence microscopy analysis of cryosections from the tissue model with fluorescently labeled monocytes [13]. The modeling of activities, resulting from different provocations in the tissue, may be particularly important in understanding mechanistic actions of adjuvants and other immunestimulatory reagents. It may also serve as a useful tool, demonstrating effects of noxious compounds in underlying clinical manifestation of lung inflammation, as well as testing reagents aimed at blocking myeloid cell activation and migration locally.
Figure 3. Relocation of DCs toward the center of the epithelial cell layer upon stimulation. (A and B) Side-view immunofluorescence images of 140 m z projections of the 3D lung-tissue models with 16HBE epithelial cells (green) and DCs (blue), in the absence (A) or presence (B) of Pam3CSK4 (1000 ng, 4 h). (C) DC migration as distance (z direction) from the center of the epithelial cell layer, 4 h poststimulation with LPS (200 ng), Pam3CSK4 (1000 ng), and CCL2 (200 ng). The analyses were based on three separate experiments. (D) Comparing the extent of DC displacement in response to 100 ng (left) and 200 ng (right) of LPS, based on the parameter track length. Each dot in the graph represents one individual DC, and data are shown with black bars as mean values from one representative experiment ⫾ sem. The statistical significance (one-way ANOVA applying Kruskal-Wallis test) of the indicated P values was determined: *P ⫽ 0.05; ***P ⬍ 0.005; ns ⫽ nonsignificant.
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To elucidate further the influence that inflammatory provocation has on DC cell motility in the lung-tissue models stimulated with LPS, Pam3CSK4, and CCL2, DC migratory behavior was monitored for 16 h. Automated tracking was used to quantitate the displacement for each individual DC (Fig. 4A and B), and the length and speed with which DCs moved under the different conditions applied were quantified. This revealed that DCs moved longer distances in combination with an increase in the mean of average cell
velocities in response to LPS, Pam3CSK4, and CCL2 (Fig. 4C and D). In addition, DCs in stimulated models exhibited a less-spherical shape (Fig. 4E) compared with unstimulated models. The morphological alteration of DCs was most predominant in models stimulated with LPS (Fig. 4E). Furthermore, a reduced spherical morphology correlated with an increase in DC mean velocity in the organotypic lung tissue model (Fig. 4F–I) and is consistent with studies showing that a prerequisite for cellular migration is that cells can
Figure 4. Quantitative analysis of DC migrating behavior in the inflammatory lung microenvironment. (A) A 3D-rendered image of a tissue model showing individual DCs (blue) only. (B) Automated colorcoded tracks of the DCs represent increasing time from start of imaging (blue) to end of imaging (red). Grid-spacing (distance between major tick marks) is 50 m for x, y, and z orientation in all images. (C–E) Comparison of the extent of DC motility with different immune-stimulating reagents, based on the parameter track length, track speed, and track sphericity. The less spherical the cells, the more their spherical measure deviates from the value 1. (F–I) Correlation graphs of track speed mean versus track sphericity mean. All results are representatives of at least three independent experiments. The statistical significance (one-way ANOVA applying Kruskal-Wallis test and correlation analysis in GraphPad Prism, v. 5) of the indicated P values was determined: *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.005, ns ⫽ nonsignificant.
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adopt an elongated morphology [19]. These data, we believe, provide new possibilities in investigating mechanisms of tissue communication circuits of the human lung tissue that underly DC motility in the microenvironment [20].
LPS stimulation of the organotypic model of the human lung induces inflammation and DC activation To verify that TLR agonist stimulations induced an inflammatory environment in the lung-tissue models, we examined the production of TNF in response to LPS and Pam3CSK at the endpoint for the live-imaging analysis (16 h poststimulation). This revealed that LPS, but not Pam3CSK4, induced detectable levels of TNF (Fig. 5A). At this time-point, no increase in TNF mRNA expression was detected in models stimulated with LPS (Fig. 5B) or Pam3CSK4 (data not shown), which is consistent with the fact that TNF is an early transient inflammatory marker, whose transcription level peaks within 1–2 h of stimulation [21]. In contrast, the CXCL8 and IL-1 mRNA tran-
scription levels were elevated in response to LPS at 16 h poststimulation (Fig. 5B), confirming that inflammation persists and may continue to affect the DC in the tissue model. However, we could not detect any difference in the CXCL8 or IL-1 mRNA levels in response to Pam3CSK4 stimulation, despite the fact that we detected enhanced DC mobility in Pam3CSK4-stimulated models (Figs. 3 and 4). The reasons for this can be time-dependent factors, but also, specific regulatory mechanisms might be involved [22], and this needs to be investigated further. To confirm that the course of inflammation induced by LPS was associated with cellular DC activation, DCs were retrieved from tissue models by enzymatic digestion, and individual cells were analyzed for the expression of relevant cell-surface markers by flow cytometry. DCs were identified as CD45⫹ cells with a relatively high surface expression of HLA-DR and a relatively low surface expression of CCR2 (Fig. 5C). At 16 h of LPS stimulation, DCs showed an increased level of surface HLA-DR and
Figure 5. Induction of inflammation and DC activation in the lung-tissue microenvironment stimulated with LPS. (A) The amount of detectable TNF in culture supernatants from tissue models stimulated with LPS or Pam3CSK4 is shown. (B) qPCR analyses of TNF, CXCL8, and IL-1 mRNA expression in unstimulated or LPS-stimulated tissue models. (C) Dot plots (dead cells excluded) and histograms (live CD45⫹ cells) from flow cytometry analyses of cells retrieved from digested tissue models and stained with a Live/Dead cell marker, in combination with anti-CD45, EpCAM HLA-DR, and CCR2 antibodies. SSC, Side-scatter; FSC, forward-scatter. (D and E) The fluorescence intensity or relative mean fluorescence intensity (MFI) of surface HLA-DR (D) and CD86 (E) on DCs (live CD45⫹ cells) retrieved from unstimulated (green) tissue models or tissue models stimulated with various concentrations of LPS, 2 ng (blue), 20 ng (black), and 200 ng (red). (D and E) The histograms represent results from one donor, whereas the bar graphs represents pooled results from six different donors and two independent experiments. The relative expression levels were calculated by subtracting the background mean fluorescence intensity (fluorescence minus one) for each sample and then dividing by the mean fluorescence intensity of the unstimulated sample for the same donor. All results are representatives of at least two independent experiments, and nontransduced 16HBE and MRC-5 ells were used for these experiments. The statistical significance (Mann-Whitney U-test in GraphPad Prism, v.5) of the indicated P values was determined: *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.005.
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CD86, consistent with cellular activation and maturation (Fig. 5D and E). Although these data demonstrated that LPS induces inflammation and DC maturation in the tissue-model system, we cannot conclude, at this stage, whether the LPS acts directly or indirectly on the DC. Nevertheless, these results demonstrate that the tissue-model system can serve as a basis for detailed investigations regarding the prerequisites for DC activation and migration in human lung epithelial tissue. In addition, this organotypic model system provides a robust and reproducible set-up appropriate to identify distinct signaling pathways in human lung tissue relevant to DC function, even beyond cellular migration and activation, in response to TLR agonists and chemokines. Robust and reliable tissue-model systems are key to advance further the field of basic human immunology, as well as the translational exploitation of such basic knowledge. Assays of human DC behavior in live tissue are generally difficult to perform, but this may be overcome by the set-up and use of tissue-model systems, as described here, which resemble the morphological and functional features of their in vivo parental tissues. Model systems should also allow intervention in combination with 4D (x, y, z, time) fluorescence imaging. This is also in line with previous studies that have shown that DC migration is largely influenced by tissue origin, degree of maturity, and the 3D structure of the microenvironment [23]. Although it is becoming evident that tissue homeostasis in the lung is dependent on epithelial cell-mediated regulation of DC [5], the precise mechanisms by which different human DC subsets are regulated during homeostasis and in response to microbial stimuli require more detailed investigations. Therefore, it is important to develop further the existing organotypic model of the human lung to allow the implantation and/or differentiation of distinct DC subsets. Potentially, strategies that involve differentiation of CD34⫹ progenitors under conditions supporting development of precursor DCs [24], which have the potential to generate distinct DC subsets, could be used for implantation into the tissue models. Comprehensive flow cytometry analyses, combined with live-imaging experiments of model tissues stimulated with compounds of distinct material and compositions, can then be applied for in-depth investigations regarding human DC differentiation, activation, and function in tissue. Thus, this model system provides a tool to identify how site-specific stimulation with particular compounds aimed at activating DCs, e.g., adjuvant and vaccine candidates and noxious compounds [25], can be exploited to provoke site-specific DC differentiation, migration, and activation. The quantification of DC redistribution and characterization of DC activation in response to inflammation, we anticipate, can also provide a powerful tool to explore, in detail, the mechanisms dictating DC anchoring and migrating behavior regulated by chemokine receptors and their ligands, present within the lung epithelial cell layer. Furthermore, this model system is also anticipated to provide a means for identifying the efficacy of anti-inflammatory reagents on DC activation during inflammation. Overall, these data demonstrate that this protocol is useful for visualizing processes of human DC migration and activation in models mimicking real tissue and that it is amendable to use with multiple stimuli. 488 Journal of Leukocyte Biology
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AUTHORSHIP A.T.N.H., P.C. and S.B. conducted the experiments. K.H. and A.G. transduced the fibroblast and epithelial cell lines with fluorescent proteins. J.G.L. and M.C. provided supervision on live-imaging data acquisition and analyses. M.S. supervised the study and contributed to the analysis and interpretation of the results. A.T.N.H. and M.S. wrote most parts of the manuscript. All authors contributed to the study design and participated in editing and finalizing the manuscript, which was approved by all authors.
ACKNOWLEDGMENTS Work in the authors’ laboratory is supported by grants from the Swedish Research Council, the Karolinska Institutet, The Knut and Alice Wallenberg Foundation, Stockholm County Council, and The Swedish Fund for Research Without Animal Experiments. P.C. and A.T.N.H. are both recipients of a Karolinska Postgraduate Studentship. This study was, in part, performed at the Live Cell Imaging Unit, Department of Biosciences and Nutrition, Karolinska Institutet (Huddinge, Sweden), supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, and the Centre for Bioscences. The authors thank Salah Zangenah for taking photos illustrating the laboratory work, as well as Aenni Ali and Sylvie Le Guyader for excellent technical assistance with cell cultures and Imaris analysis, respectively.
DISCLOSURES
No conflict of interest, financial or otherwise, is declared by the authors.
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KEY WORDS: 3D-tissue models 䡠 epithelial tissue 䡠 Toll-like receptor agonists 䡠 inflammation
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