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Study of dendritic cell migration using micro-fabrication
JIM-12117; No of Pages 5 Journal of Immunological Methods xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Study of dendritic cell migration using micro-fabrication Pablo Vargas a,⁎, Mélanie Chabaud b,1, Hawa-Racine Thiam a,2, Danielle Lankar b, Matthieu Piel a, Ana-Maria Lennon-Dumenil b a b
Institut Curie, CNRS UMR 144, 26 rue d'Ulm, 75005 Paris, France Institut Curie, Inserm U932, 26 rue d'Ulm, 75005 Paris, France
a r t i c l e
i n f o
Article history: Received 17 August 2015 Received in revised form 13 November 2015 Accepted 8 December 2015 Available online xxxx Keywords: Cell migration Motility Dendritic cells Lymphocytes Leukocytes Confinement Micro-fabrication Imaging Chemokines Gradients Constrictions Persistence Cell polarity
a b s t r a c t Cell migration is a hallmark of dendritic cells (DCs) function. It is needed for DCs to scan their environment in search for antigens as well as to reach lymphatic organs in order to trigger T lymphocyte's activation. Such interaction leads to tolerance in the case of DCs migrating under homeostatic conditions or to immunity in the case of DCs migrating upon encounter with pathogen-associated molecular patterns. Cell migration is therefore essential for DCs to transfer information from peripheral tissues to lymphoid organs, thereby linking innate to adaptive immunity. This stresses the need to unravel the molecular mechanisms involved. However, the tremendous complexity of the tissue microenvironment as well as the limited spatio-temporal resolution of in vivo imaging techniques has made this task difficult. To bypass this problem, we have developed microfabrication-based experimental tools that are compatible with high-resolution imaging. Here, we will discuss how such devices can be used to study DC migration under controlled conditions that mimic their physiological environment in a robust quantitative manner. © 2015 Published by Elsevier B.V.
1. Introduction Cell migration plays a key role in immune responses. At early stages of infection it allows pathogen clearance by promoting the local concentration of phagocytes at the site of inflammation (Soehnlein and Lindbom, 2010). Later, during the adaptive immunity response, a more complex migration program between distant locations is established. This program is characterized by exchanges of cells between infected tissues and lymphoid organs, a migration game intended to promote communication between distant compartments to ensure both tolerance to self and elimination of infectious agents. In that context, DCs play a key role in transporting antigens to lymph nodes for presentation to T lymphocytes either under homeostatic conditions for establishment of tolerance, or during infection for the onset of the immune response (Baratin et al., 2015).
⁎ Corresponding author. E-mail address: [email protected] (P. Vargas). Current address: Centre for Vascular Research and Australian Centre for NanoMedicine, University of New South Wales, Sydney 2052, Australia. 2 Current address: Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. 1
DC migration from peripheral tissues to draining lymph nodes occurs through lymphatic vessels (LVs) and requires the upregulation of the chemokine receptor CCR7, which binds both CCL19 and CCL21 upon DC activation. In the mouse skin, CCL21 produced by lymphatic endothelial cells forms an immobilized gradient that is followed by activated DCs and is required for their recruitment to LVs (Weber et al., 2013). To enter these lymphatics DCs deform and expand preexisting portals localized at their surface (Pflicke and Sixt, 2009). At this stage, the expression of Podoplanin at the surface of endothelial cells helps recruiting mature DCs into LVs (Acton et al., 2012). In lymph nodes, CCL21 is produced by fibroblastic reticular cells (Link et al., 2007) at the subcapsular sinus and locally scavenged by its non-signaling receptor CCRL1 (Ulvmar et al., 2014). This allows gradient formation and penetration of DCs to the lymph node T cell zone. The ability of tissue-resident immature DCs to explore their environment searching for harmful agents was shown to be associated to active motility in both the gut and the skin (Farache et al., 2013; Ng et al., 2008). Although this occurs independently of CCR7, it has been shown to involve the chemokine receptor CX3CR1 and its ligand Fractalkine or CX3CL1 in the gut, which is produced by epithelial cells (Niess et al., 2005; Kim et al., 2011). In addition, chemokine receptors such as CCR5 were shown to be down-regulated at the surface of DCs upon activation by inflammatory stimuli (Le et al., 2001).
http://dx.doi.org/10.1016/j.jim.2015.12.005 0022-1759/© 2015 Published by Elsevier B.V.
Please cite this article as: Vargas, P., et al., Study of dendritic cell migration using micro-fabrication, J. Immunol. Methods (2015), http://dx.doi.org/ 10.1016/j.jim.2015.12.005
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P. Vargas et al. / Journal of Immunological Methods xxx (2015) xxx–xxx
Fig. 1. DCs migration in micro-fabricated channels A. Scheme of a micro-fabricated device used to evaluate cell speed. DCs are loaded in the “loading chamber” and then spontaneously migrate in the micro-channels (inset). B. Scanning electron microscopy of an immature DC in a micro-channel. C. Time lapse of an immature DC migrating in a micro-channel. Fast and slow motility phases can be observed.
However, whether the corresponding ligands form gradients in vivo and thereby influence environment patrolling by immature DCs remains to be established. Furthermore, the molecular mechanisms by which DCs sense and respond to chemokine gradients by modifying their migration capacity are far from being fully understood. This results at least in part by the tremendous complexity of tissues as well as by the limited resolution of in vivo imaging techniques. In addition to rely on the external biochemical cues described above, DC migration is strongly dependent on physical properties of their environment. In particular, their motility is highly sensitive to external geometry as illustrated by their capacity to move in 3- but not 2-dimensional environments in the absence of integrins or to migrate significantly faster when confined (Lammermann et al., 2008; Heuzé et al., 2013). This stresses the need to develop experimental systems that are compatible with high-resolution microscopy but can also be used to isolate specific biochemical or physical properties of tissues in order to define their impact on the migratory capacity of DCs. Here, we review the use of confined micro-fabricated channels to study, in a controlled environment, different aspects of DC migration and dissect the underlying molecular mechanisms. 2. Generation of micro-fabricated channels to study spontaneous DC migration In general, when migrating in tissues, cells are immersed in a complex landscape composed of extracellular matrix and other cells.
This configuration can vary depending on the organ, but retains a common property: cells migrate in constrained environments. To mimic this property of tissues we use micro-channels, tiny tunnels in which cells migrate under confinement. To make these channels, PDMS, a polymer permeable to gas that allows sample oxygenation, is poured into micro-fabricated molds. These are used to generate large sets of chips with identical geometries. Starting from a mold, preparation of micro-channels is fast, simple and inexpensive (Vargas et al., 2014). Channels exhibit a square or rectangular section and their size and shape can be defined based on which geometrical property of the cell environment one wishes to isolate (see Section 3). Three channel walls made of PDMS are stuck on a glass coverslip, making these disposable devices compatible with high-resolution imaging (Fig. 1A). They can be coated with extracellular matrix protein such as fibronectin or collagen. Cells are loaded into a central chamber communicated with multiple micro-channels that can be analyzed simultaneously by microscopy (Fig. 1A). This allows the generation of large datasets from each experiment from which key migration parameters such as cell speed and directionality are automatically quantified. This is achieved either by using state-of-the-art software to track the nucleus of Hoechst-labeled cells, or by generating kymographs from processed phase contrast images (Vargas et al., 2014). Importantly, each migration experiment can be done with few thousands of cells, making it compatible with mouse or human primary samples (Fernandez et al., 2011). As mentioned above, tissue scanning by immature DCs can be associated to active cell migration. Because immature DCs spontaneously
Please cite this article as: Vargas, P., et al., Study of dendritic cell migration using micro-fabrication, J. Immunol. Methods (2015), http://dx.doi.org/ 10.1016/j.jim.2015.12.005
P. Vargas et al. / Journal of Immunological Methods xxx (2015) xxx–xxx
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enter and migrate in micro-fabricated channels, we have used such device to study fundamental aspects of immature DC motility (FaureAndre et al., 2008). Indeed, the length of micro-channels can be tuned to monitor cell migration over long distances, allowing the study of DC motility either under homeostatic conditions or in response to extracellular stimuli. Using 4 μm (height) × 5 μm (wide) micro-channels, we showed that immature DCs differentiated from mouse bone marrow migrate with a mean velocity of ~6 μm/min (Faure-Andre et al., 2008), a value that is similar to the speed they reach in tissues (Lammermann et al., 2008), validating the use of this device to study their locomotion properties in vitro. More importantly, the use of micro-channels led to an unexpected finding: immature DCs use a peculiar locomotion mode that alternates phases of rapid migration with phases of arrest (Faure-Andre et al., 2008; Chabaud et al., 2015). It would have not been possible to highlight such periodic pauses by using methods to study DC migration in classical 2-dimensional devices or in tissues as cells would have changed their direction of movement while decreasing their speed. This highlights how micro-fabricated tools that allow the control of specific parameters of the cell environment can be valuable to unravel the cell-intrinsic component of DC locomotion. Of note, an additional advantage of micro-channels is that they allow naturally selected motile cells among a mixed cell population. This is particularly useful in the context of bone-marrow derived cell cultures since it allows separating immature DCs, which are migratory cells, from contaminant macrophages, which are sticky and non-motile (Helft et al., 2015). 3. Using micro-channels to study organelle dynamics during DC migration Beyond their compatibility with cell imaging at high spatial and temporal resolution, micro-channels present the advantage of forcing cells to adopt a polarized elongated shape thereby normalizing cell morphology among the population (Fig. 1B and C). This allows visualizing how specific subcellular structures evolve during cell motility and facilitate the dissection of the molecular mechanisms that govern DCs migration. One of the first observations we made thanks to this property of microchannels was the presence of giant vesicles at the front of immature cells, which formed while the cell moves forward and were resorbed during arrest phases (Fig. 2A and B). Further studies showed that they corresponded to macropinosomes that are used by immature DCs to internalize extracellular material (Chabaud et al., 2015). The dynamics of these vesicles in terms of size, number and lifetime could be analyzed while DCs migrated in micro-channels by adding a fluorescent protein into the extracellular medium (Fig. 2A, lower panel) (Chabaud et al., 2015). When using proteins labeled with both a pH-insensitive and a pH-sensitive dye, the transport of antigens from macropinosomes to acidic endolysosomes – where they are processed for presentation to T cells – could be quantified (Chabaud et al., 2015). In migrating DCs, endolysosomes were usually observed at the cell back, physically separated by the nucleus (Fig. 2A). These results illustrate that the polarized morphology that cells adopt in micro-channels helps spatially separating their organelles, providing a unique system to quantify their dynamics during cell migration. 4. Using micro-channels to study protein and calcium dynamics during DC migration In addition to intracellular compartments, micro-channels can be used to visualize the intracellular distribution of fluorescent proteins during cell motility. For example, using Myosin IIA-GFP knock in DCs, we were able to analyze the dynamics of this actin-based motor protein in migrating DCs (Chabaud et al., 2015; Solanes et al., 2015). Quantitative analysis of the distribution of Myosin IIA-GFP on a large number of cells showed that it accumulated at the rear of immature DCs while they were in fast motility phases but was recruited to their front during
Fig. 2. High resolution imaging of DCs migrating in micro-channels A. Membrane staining (green) and internalized material (red) of an immature DC migrating in a micro-channel. Picture was taken using a spinning disk microscope (100×). Zone of micropinocytosis (front) and lysosomes (back) are highlighted. B. LifeAct-GFP DC migrating in micro-channels time-lapsed on a spinning disk microscope (100×) every 400 ms. Macropinosomes dynamics can be observed at the front of the migrating DC. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
slow motion (Chabaud et al., 2015). Similarly, F-actin was enriched around the macropinosomes that formed at the front of migrating immature DCs allowing the visualization of local vesicle fission and fusion events (Fig. 2B). The asymmetric distribution of organelles and molecules raised the question of whether they differentially contribute to the physiology of DCs. To address this question, micro-channels can be used used to deliver drugs either at the front or at the rear of migrating cells (Chabaud et al., 2015). This method allowed us dissecting the specific roles played by Myosin II at both subcellular locations. We found that the pool of Myosin II located at the back of the cell was responsible for fast locomotion whereas its recruitment to the cell front decreased cell speed but promoted antigen capture by macropinocytosis. We further showed that diversion of Myosin II from the DC rear to the DC front relied on its interaction with the MHC class II-associated Invariant Chain (CD74). These results allowed us to build a physical model based on intermittent search strategies suggesting that the bimodal mode of DC migration imposed by CD74 optimizes their environment patrolling capacity (Chabaud et al., 2015). Finally, micro-channels can also be used to correlate key DC migration parameters such as cell speed and movement directionality with intracellular calcium dynamics. This can be achieved by imaging migration of cells loaded with fluorescent calcium-sensitive dyes (Solanes et al., 2015). By performing this analysis, we made several important observations on the role of this second messenger on DC migration. First, we found that intracellular calcium stores were released from the endoplasmic reticulum during phases of fast motility. Second, we observed that these events of calcium release were required for DCs to maintain Myosin IIA polarity so that they do not lose their directionality after
Please cite this article as: Vargas, P., et al., Study of dendritic cell migration using micro-fabrication, J. Immunol. Methods (2015), http://dx.doi.org/ 10.1016/j.jim.2015.12.005
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Fig. 3. Micro-channels to study the impact of extracellular cues on DC migration A. Time lapse of an immature DC migrating through a constriction (15 μm length). Cell and nucleus deformation while passing by the narrow space can be observed. B. Mean speed of activated DCs migrating in channels coated homogeneously with different doses of CCL21.
slow motility phases and can therefore better explore the extracellular space (Solanes et al., 2015). 5. Using micro-channels to study the impact of extracellular cues on DC migration In tissues, DC migration is regulated by both the biochemical and physical properties of their microenvironment. In general, microfabrication provides a unique tool to address the contribution of isolated environmental cues to the cell migratory behavior. Indeed, an essential aspect of micro-fabrication is that it supports the construction of channels of different shapes and coated with different extracellular matrix proteins. For example, cell plasticity can be challenged by reducing the size of the channels during migration (Fig. 3A). Introduction of these “constrictions” forces cells to encounter a physical barrier during locomotion (Heuze et al., 2011). We used this system to study the mechanisms that allow DCs squeezing themselves in order to migrate through small holes in vivo, in particular during cell intravasation (Pflicke and Sixt, 2009; Wolf et al., 2013). We observed that DCs were able to flatten their cell body in order to cross channel constrictions, highlighting their incredible deformation capacity (Fig. 3A). Such peculiarity of DCs could be further dissected by building constrictions of distinct width and length or by analyzing how the components that control the mechanical properties of DCs alter their ability to cross these small holes. Additional variation in the shape of micro-channels includes the introduction of bifurcations that can be used to unravel how cells chose a direction when experiencing several possible choices (Prentice-Mott et al., 2013). As mentioned above, DC migration in tissues also responds to guidance by extracellular chemokines. Although some of these molecules are produced in a soluble manner, it was recently shown that others such as CCL21 in the mouse skin or CX3CL1 in the mouse gut are modified so that they bind to extracellular matrix components (Weber et al., 2013; Kim et al., 2011). The existence of such “haptotactic chemokine gradients” has only been demonstrated in the mouse skin in vivo (Weber et al., 2013). In addition, how DCs recognize these immobilized chemokine gradients has not been established. Micro-channels can be used to study the haptotactic migration of DCs by coating the channel walls with chemokines. Functionalization of micro-channels with increasing doses of CCL21 triggers a dose-dependent increase of the DC migration speed (Fig. 3B). These results demonstrate that DCs sense and respond to immobilized CCL21 by migrating faster. Micro-fluidic systems such as the one described above for selective drug delivery at
one cell pole could also be used to generate gradients of chemoattractants or of other extracellular molecule such as Podoplanin along micro-channels. 6. Conclusions Migration of DCs in tissues is a complex multi-factorial process that is thus difficult to study in their natural environment. Micro-fabrication offers the possibility to develop robust bottom-up approaches that allow evaluating the specific contribution of individual physical and biochemical extracellular cues to DC migration as well as to dissecting the underlying molecular mechanisms. They therefore represent a valuable controlled experimental system from which hypotheses to be later tested in vivo can be generated. Although the design of channels of different size is defined by the resolution limits of micro-fabrication, their possible shapes are only restricted to the creativity of the user. Control of the environment geometry, of the channel coating, compatibility with highresolution microscopy and the generation of large sets of quantifiable data under distinct experimental conditions make these tools unique for the general study of immune cell migration. References Acton, S.E., et al., 2012. Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2. Immunity 37 (2), 276–289. Baratin, M., et al., 2015. Homeostatic NF-kappaB signaling in steady-state migratory dendritic cells regulates immune homeostasis and tolerance. Immunity 42 (4), 627–639. Chabaud, M., et al., 2015. Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells. Nat. Commun. 6, 7526. Farache, J., et al., 2013. Luminal bacteria recruit CD103+ dendritic cells into the intestinal epithelium to sample bacterial antigens for presentation. Immunity 38 (3), 581–595. Faure-Andre, G., et al., 2008. Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain. Science 322 (5908), 1705–1710. Fernandez, M.I., et al., 2011. The human cytokine TSLP triggers a cell-autonomous dendritic cell migration in confined environments. Blood 118 (14), 3862–3869. Helft, J., et al., 2015. GM-CSF mouse bone marrow cultures comprise a heterogeneous population of CD11c(+)MHCII(+) macrophages and dendritic cells. Immunity 42 (6), 1197–1211. Heuze, M.L., et al., 2011. Cell migration in confinement: a micro-channel-based assay. Methods Mol. Biol. 769, 415–434. Heuzé, M.L., et al., 2013. Migration of dendritic cells: physical principles, molecular mechanisms, and functional implications. Immunol. Rev. 256 (1), 240–254. Kim, K.W., et al., 2011. In vivo structure/function and expression analysis of the CX3C chemokine fractalkine. Blood 118 (22), e156–e167. Lammermann, T., et al., 2008. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453 (7191), 51–55. Le, Y., et al., 2001. Desensitization of chemokine receptor CCR5 in dendritic cells at the early stage of differentiation by activation of formyl peptide receptors. Clin. Immunol. 99 (3), 365–372.
Please cite this article as: Vargas, P., et al., Study of dendritic cell migration using micro-fabrication, J. Immunol. Methods (2015), http://dx.doi.org/ 10.1016/j.jim.2015.12.005
P. Vargas et al. / Journal of Immunological Methods xxx (2015) xxx–xxx Link, A., et al., 2007. Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells. Nat. Immunol. 8 (11), 1255–1265. Ng, L.G., et al., 2008. Migratory dermal dendritic cells act as rapid sensors of protozoan parasites. PLoS Pathog. 4 (11), e1000222. Niess, J.H., et al., 2005. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 307 (5707), 254–258. Pflicke, H., Sixt, M., 2009. Preformed portals facilitate dendritic cell entry into afferent lymphatic vessels. J. Exp. Med. 206 (13), 2925–2935. Prentice-Mott, H.V., et al., 2013. Biased migration of confined neutrophil-like cells in asymmetric hydraulic environments. Proc. Natl. Acad. Sci. U. S. A. 110 (52), 21006–21011. Soehnlein, O., Lindbom, L., 2010. Phagocyte partnership during the onset and resolution of inflammation. Nat. Rev. Immunol. 10 (6), 427–439.
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Solanes, P., et al., 2015. Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1. EMBO J. 34 (6), 798–810. Ulvmar, M.H., et al., 2014. The atypical chemokine receptor CCRL1 shapes functional CCL21 gradients in lymph nodes. Nat. Immunol. 15 (7), 623–630. Vargas, P., et al., 2014. Study of cell migration in microfabricated channels. J. Vis. Exp. (84), e51099. Weber, M., et al., 2013. Interstitial dendritic cell guidance by haptotactic chemokine gradients. Science 339 (6117), 328–332. Wolf, K., et al., 2013. Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force. J. Cell Biol. 201 (7), 1069–1084.
Please cite this article as: Vargas, P., et al., Study of dendritic cell migration using micro-fabrication, J. Immunol. Methods (2015), http://dx.doi.org/ 10.1016/j.jim.2015.12.005
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Study of dendritic cell migration using micro-fabrication Pablo Vargas a,⁎, Mélanie Chabaud b,1, Hawa-Racine Thiam a,2, Danielle Lankar b, Matthieu Piel a, Ana-Maria Lennon-Dumenil b a b
Institut Curie, CNRS UMR 144, 26 rue d'Ulm, 75005 Paris, France Institut Curie, Inserm U932, 26 rue d'Ulm, 75005 Paris, France
a r t i c l e
i n f o
Article history: Received 17 August 2015 Received in revised form 13 November 2015 Accepted 8 December 2015 Available online xxxx Keywords: Cell migration Motility Dendritic cells Lymphocytes Leukocytes Confinement Micro-fabrication Imaging Chemokines Gradients Constrictions Persistence Cell polarity
a b s t r a c t Cell migration is a hallmark of dendritic cells (DCs) function. It is needed for DCs to scan their environment in search for antigens as well as to reach lymphatic organs in order to trigger T lymphocyte's activation. Such interaction leads to tolerance in the case of DCs migrating under homeostatic conditions or to immunity in the case of DCs migrating upon encounter with pathogen-associated molecular patterns. Cell migration is therefore essential for DCs to transfer information from peripheral tissues to lymphoid organs, thereby linking innate to adaptive immunity. This stresses the need to unravel the molecular mechanisms involved. However, the tremendous complexity of the tissue microenvironment as well as the limited spatio-temporal resolution of in vivo imaging techniques has made this task difficult. To bypass this problem, we have developed microfabrication-based experimental tools that are compatible with high-resolution imaging. Here, we will discuss how such devices can be used to study DC migration under controlled conditions that mimic their physiological environment in a robust quantitative manner. © 2015 Published by Elsevier B.V.
1. Introduction Cell migration plays a key role in immune responses. At early stages of infection it allows pathogen clearance by promoting the local concentration of phagocytes at the site of inflammation (Soehnlein and Lindbom, 2010). Later, during the adaptive immunity response, a more complex migration program between distant locations is established. This program is characterized by exchanges of cells between infected tissues and lymphoid organs, a migration game intended to promote communication between distant compartments to ensure both tolerance to self and elimination of infectious agents. In that context, DCs play a key role in transporting antigens to lymph nodes for presentation to T lymphocytes either under homeostatic conditions for establishment of tolerance, or during infection for the onset of the immune response (Baratin et al., 2015).
⁎ Corresponding author. E-mail address: [email protected] (P. Vargas). Current address: Centre for Vascular Research and Australian Centre for NanoMedicine, University of New South Wales, Sydney 2052, Australia. 2 Current address: Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. 1
DC migration from peripheral tissues to draining lymph nodes occurs through lymphatic vessels (LVs) and requires the upregulation of the chemokine receptor CCR7, which binds both CCL19 and CCL21 upon DC activation. In the mouse skin, CCL21 produced by lymphatic endothelial cells forms an immobilized gradient that is followed by activated DCs and is required for their recruitment to LVs (Weber et al., 2013). To enter these lymphatics DCs deform and expand preexisting portals localized at their surface (Pflicke and Sixt, 2009). At this stage, the expression of Podoplanin at the surface of endothelial cells helps recruiting mature DCs into LVs (Acton et al., 2012). In lymph nodes, CCL21 is produced by fibroblastic reticular cells (Link et al., 2007) at the subcapsular sinus and locally scavenged by its non-signaling receptor CCRL1 (Ulvmar et al., 2014). This allows gradient formation and penetration of DCs to the lymph node T cell zone. The ability of tissue-resident immature DCs to explore their environment searching for harmful agents was shown to be associated to active motility in both the gut and the skin (Farache et al., 2013; Ng et al., 2008). Although this occurs independently of CCR7, it has been shown to involve the chemokine receptor CX3CR1 and its ligand Fractalkine or CX3CL1 in the gut, which is produced by epithelial cells (Niess et al., 2005; Kim et al., 2011). In addition, chemokine receptors such as CCR5 were shown to be down-regulated at the surface of DCs upon activation by inflammatory stimuli (Le et al., 2001).
http://dx.doi.org/10.1016/j.jim.2015.12.005 0022-1759/© 2015 Published by Elsevier B.V.
Please cite this article as: Vargas, P., et al., Study of dendritic cell migration using micro-fabrication, J. Immunol. Methods (2015), http://dx.doi.org/ 10.1016/j.jim.2015.12.005
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P. Vargas et al. / Journal of Immunological Methods xxx (2015) xxx–xxx
Fig. 1. DCs migration in micro-fabricated channels A. Scheme of a micro-fabricated device used to evaluate cell speed. DCs are loaded in the “loading chamber” and then spontaneously migrate in the micro-channels (inset). B. Scanning electron microscopy of an immature DC in a micro-channel. C. Time lapse of an immature DC migrating in a micro-channel. Fast and slow motility phases can be observed.
However, whether the corresponding ligands form gradients in vivo and thereby influence environment patrolling by immature DCs remains to be established. Furthermore, the molecular mechanisms by which DCs sense and respond to chemokine gradients by modifying their migration capacity are far from being fully understood. This results at least in part by the tremendous complexity of tissues as well as by the limited resolution of in vivo imaging techniques. In addition to rely on the external biochemical cues described above, DC migration is strongly dependent on physical properties of their environment. In particular, their motility is highly sensitive to external geometry as illustrated by their capacity to move in 3- but not 2-dimensional environments in the absence of integrins or to migrate significantly faster when confined (Lammermann et al., 2008; Heuzé et al., 2013). This stresses the need to develop experimental systems that are compatible with high-resolution microscopy but can also be used to isolate specific biochemical or physical properties of tissues in order to define their impact on the migratory capacity of DCs. Here, we review the use of confined micro-fabricated channels to study, in a controlled environment, different aspects of DC migration and dissect the underlying molecular mechanisms. 2. Generation of micro-fabricated channels to study spontaneous DC migration In general, when migrating in tissues, cells are immersed in a complex landscape composed of extracellular matrix and other cells.
This configuration can vary depending on the organ, but retains a common property: cells migrate in constrained environments. To mimic this property of tissues we use micro-channels, tiny tunnels in which cells migrate under confinement. To make these channels, PDMS, a polymer permeable to gas that allows sample oxygenation, is poured into micro-fabricated molds. These are used to generate large sets of chips with identical geometries. Starting from a mold, preparation of micro-channels is fast, simple and inexpensive (Vargas et al., 2014). Channels exhibit a square or rectangular section and their size and shape can be defined based on which geometrical property of the cell environment one wishes to isolate (see Section 3). Three channel walls made of PDMS are stuck on a glass coverslip, making these disposable devices compatible with high-resolution imaging (Fig. 1A). They can be coated with extracellular matrix protein such as fibronectin or collagen. Cells are loaded into a central chamber communicated with multiple micro-channels that can be analyzed simultaneously by microscopy (Fig. 1A). This allows the generation of large datasets from each experiment from which key migration parameters such as cell speed and directionality are automatically quantified. This is achieved either by using state-of-the-art software to track the nucleus of Hoechst-labeled cells, or by generating kymographs from processed phase contrast images (Vargas et al., 2014). Importantly, each migration experiment can be done with few thousands of cells, making it compatible with mouse or human primary samples (Fernandez et al., 2011). As mentioned above, tissue scanning by immature DCs can be associated to active cell migration. Because immature DCs spontaneously
Please cite this article as: Vargas, P., et al., Study of dendritic cell migration using micro-fabrication, J. Immunol. Methods (2015), http://dx.doi.org/ 10.1016/j.jim.2015.12.005
P. Vargas et al. / Journal of Immunological Methods xxx (2015) xxx–xxx
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enter and migrate in micro-fabricated channels, we have used such device to study fundamental aspects of immature DC motility (FaureAndre et al., 2008). Indeed, the length of micro-channels can be tuned to monitor cell migration over long distances, allowing the study of DC motility either under homeostatic conditions or in response to extracellular stimuli. Using 4 μm (height) × 5 μm (wide) micro-channels, we showed that immature DCs differentiated from mouse bone marrow migrate with a mean velocity of ~6 μm/min (Faure-Andre et al., 2008), a value that is similar to the speed they reach in tissues (Lammermann et al., 2008), validating the use of this device to study their locomotion properties in vitro. More importantly, the use of micro-channels led to an unexpected finding: immature DCs use a peculiar locomotion mode that alternates phases of rapid migration with phases of arrest (Faure-Andre et al., 2008; Chabaud et al., 2015). It would have not been possible to highlight such periodic pauses by using methods to study DC migration in classical 2-dimensional devices or in tissues as cells would have changed their direction of movement while decreasing their speed. This highlights how micro-fabricated tools that allow the control of specific parameters of the cell environment can be valuable to unravel the cell-intrinsic component of DC locomotion. Of note, an additional advantage of micro-channels is that they allow naturally selected motile cells among a mixed cell population. This is particularly useful in the context of bone-marrow derived cell cultures since it allows separating immature DCs, which are migratory cells, from contaminant macrophages, which are sticky and non-motile (Helft et al., 2015). 3. Using micro-channels to study organelle dynamics during DC migration Beyond their compatibility with cell imaging at high spatial and temporal resolution, micro-channels present the advantage of forcing cells to adopt a polarized elongated shape thereby normalizing cell morphology among the population (Fig. 1B and C). This allows visualizing how specific subcellular structures evolve during cell motility and facilitate the dissection of the molecular mechanisms that govern DCs migration. One of the first observations we made thanks to this property of microchannels was the presence of giant vesicles at the front of immature cells, which formed while the cell moves forward and were resorbed during arrest phases (Fig. 2A and B). Further studies showed that they corresponded to macropinosomes that are used by immature DCs to internalize extracellular material (Chabaud et al., 2015). The dynamics of these vesicles in terms of size, number and lifetime could be analyzed while DCs migrated in micro-channels by adding a fluorescent protein into the extracellular medium (Fig. 2A, lower panel) (Chabaud et al., 2015). When using proteins labeled with both a pH-insensitive and a pH-sensitive dye, the transport of antigens from macropinosomes to acidic endolysosomes – where they are processed for presentation to T cells – could be quantified (Chabaud et al., 2015). In migrating DCs, endolysosomes were usually observed at the cell back, physically separated by the nucleus (Fig. 2A). These results illustrate that the polarized morphology that cells adopt in micro-channels helps spatially separating their organelles, providing a unique system to quantify their dynamics during cell migration. 4. Using micro-channels to study protein and calcium dynamics during DC migration In addition to intracellular compartments, micro-channels can be used to visualize the intracellular distribution of fluorescent proteins during cell motility. For example, using Myosin IIA-GFP knock in DCs, we were able to analyze the dynamics of this actin-based motor protein in migrating DCs (Chabaud et al., 2015; Solanes et al., 2015). Quantitative analysis of the distribution of Myosin IIA-GFP on a large number of cells showed that it accumulated at the rear of immature DCs while they were in fast motility phases but was recruited to their front during
Fig. 2. High resolution imaging of DCs migrating in micro-channels A. Membrane staining (green) and internalized material (red) of an immature DC migrating in a micro-channel. Picture was taken using a spinning disk microscope (100×). Zone of micropinocytosis (front) and lysosomes (back) are highlighted. B. LifeAct-GFP DC migrating in micro-channels time-lapsed on a spinning disk microscope (100×) every 400 ms. Macropinosomes dynamics can be observed at the front of the migrating DC. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
slow motion (Chabaud et al., 2015). Similarly, F-actin was enriched around the macropinosomes that formed at the front of migrating immature DCs allowing the visualization of local vesicle fission and fusion events (Fig. 2B). The asymmetric distribution of organelles and molecules raised the question of whether they differentially contribute to the physiology of DCs. To address this question, micro-channels can be used used to deliver drugs either at the front or at the rear of migrating cells (Chabaud et al., 2015). This method allowed us dissecting the specific roles played by Myosin II at both subcellular locations. We found that the pool of Myosin II located at the back of the cell was responsible for fast locomotion whereas its recruitment to the cell front decreased cell speed but promoted antigen capture by macropinocytosis. We further showed that diversion of Myosin II from the DC rear to the DC front relied on its interaction with the MHC class II-associated Invariant Chain (CD74). These results allowed us to build a physical model based on intermittent search strategies suggesting that the bimodal mode of DC migration imposed by CD74 optimizes their environment patrolling capacity (Chabaud et al., 2015). Finally, micro-channels can also be used to correlate key DC migration parameters such as cell speed and movement directionality with intracellular calcium dynamics. This can be achieved by imaging migration of cells loaded with fluorescent calcium-sensitive dyes (Solanes et al., 2015). By performing this analysis, we made several important observations on the role of this second messenger on DC migration. First, we found that intracellular calcium stores were released from the endoplasmic reticulum during phases of fast motility. Second, we observed that these events of calcium release were required for DCs to maintain Myosin IIA polarity so that they do not lose their directionality after
Please cite this article as: Vargas, P., et al., Study of dendritic cell migration using micro-fabrication, J. Immunol. Methods (2015), http://dx.doi.org/ 10.1016/j.jim.2015.12.005
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Fig. 3. Micro-channels to study the impact of extracellular cues on DC migration A. Time lapse of an immature DC migrating through a constriction (15 μm length). Cell and nucleus deformation while passing by the narrow space can be observed. B. Mean speed of activated DCs migrating in channels coated homogeneously with different doses of CCL21.
slow motility phases and can therefore better explore the extracellular space (Solanes et al., 2015). 5. Using micro-channels to study the impact of extracellular cues on DC migration In tissues, DC migration is regulated by both the biochemical and physical properties of their microenvironment. In general, microfabrication provides a unique tool to address the contribution of isolated environmental cues to the cell migratory behavior. Indeed, an essential aspect of micro-fabrication is that it supports the construction of channels of different shapes and coated with different extracellular matrix proteins. For example, cell plasticity can be challenged by reducing the size of the channels during migration (Fig. 3A). Introduction of these “constrictions” forces cells to encounter a physical barrier during locomotion (Heuze et al., 2011). We used this system to study the mechanisms that allow DCs squeezing themselves in order to migrate through small holes in vivo, in particular during cell intravasation (Pflicke and Sixt, 2009; Wolf et al., 2013). We observed that DCs were able to flatten their cell body in order to cross channel constrictions, highlighting their incredible deformation capacity (Fig. 3A). Such peculiarity of DCs could be further dissected by building constrictions of distinct width and length or by analyzing how the components that control the mechanical properties of DCs alter their ability to cross these small holes. Additional variation in the shape of micro-channels includes the introduction of bifurcations that can be used to unravel how cells chose a direction when experiencing several possible choices (Prentice-Mott et al., 2013). As mentioned above, DC migration in tissues also responds to guidance by extracellular chemokines. Although some of these molecules are produced in a soluble manner, it was recently shown that others such as CCL21 in the mouse skin or CX3CL1 in the mouse gut are modified so that they bind to extracellular matrix components (Weber et al., 2013; Kim et al., 2011). The existence of such “haptotactic chemokine gradients” has only been demonstrated in the mouse skin in vivo (Weber et al., 2013). In addition, how DCs recognize these immobilized chemokine gradients has not been established. Micro-channels can be used to study the haptotactic migration of DCs by coating the channel walls with chemokines. Functionalization of micro-channels with increasing doses of CCL21 triggers a dose-dependent increase of the DC migration speed (Fig. 3B). These results demonstrate that DCs sense and respond to immobilized CCL21 by migrating faster. Micro-fluidic systems such as the one described above for selective drug delivery at
one cell pole could also be used to generate gradients of chemoattractants or of other extracellular molecule such as Podoplanin along micro-channels. 6. Conclusions Migration of DCs in tissues is a complex multi-factorial process that is thus difficult to study in their natural environment. Micro-fabrication offers the possibility to develop robust bottom-up approaches that allow evaluating the specific contribution of individual physical and biochemical extracellular cues to DC migration as well as to dissecting the underlying molecular mechanisms. They therefore represent a valuable controlled experimental system from which hypotheses to be later tested in vivo can be generated. Although the design of channels of different size is defined by the resolution limits of micro-fabrication, their possible shapes are only restricted to the creativity of the user. Control of the environment geometry, of the channel coating, compatibility with highresolution microscopy and the generation of large sets of quantifiable data under distinct experimental conditions make these tools unique for the general study of immune cell migration. References Acton, S.E., et al., 2012. Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2. Immunity 37 (2), 276–289. Baratin, M., et al., 2015. Homeostatic NF-kappaB signaling in steady-state migratory dendritic cells regulates immune homeostasis and tolerance. Immunity 42 (4), 627–639. Chabaud, M., et al., 2015. Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells. Nat. Commun. 6, 7526. Farache, J., et al., 2013. Luminal bacteria recruit CD103+ dendritic cells into the intestinal epithelium to sample bacterial antigens for presentation. Immunity 38 (3), 581–595. Faure-Andre, G., et al., 2008. Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain. Science 322 (5908), 1705–1710. Fernandez, M.I., et al., 2011. The human cytokine TSLP triggers a cell-autonomous dendritic cell migration in confined environments. Blood 118 (14), 3862–3869. Helft, J., et al., 2015. GM-CSF mouse bone marrow cultures comprise a heterogeneous population of CD11c(+)MHCII(+) macrophages and dendritic cells. Immunity 42 (6), 1197–1211. Heuze, M.L., et al., 2011. Cell migration in confinement: a micro-channel-based assay. Methods Mol. Biol. 769, 415–434. Heuzé, M.L., et al., 2013. Migration of dendritic cells: physical principles, molecular mechanisms, and functional implications. Immunol. Rev. 256 (1), 240–254. Kim, K.W., et al., 2011. In vivo structure/function and expression analysis of the CX3C chemokine fractalkine. Blood 118 (22), e156–e167. Lammermann, T., et al., 2008. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453 (7191), 51–55. Le, Y., et al., 2001. Desensitization of chemokine receptor CCR5 in dendritic cells at the early stage of differentiation by activation of formyl peptide receptors. Clin. Immunol. 99 (3), 365–372.
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Please cite this article as: Vargas, P., et al., Study of dendritic cell migration using micro-fabrication, J. Immunol. Methods (2015), http://dx.doi.org/ 10.1016/j.jim.2015.12.005