Letter pubs.acs.org/NanoLett
Carbon Nanotube Scaffolds Instruct Human Dendritic Cells: Modulating Immune Responses by Contacts at the Nanoscale Alessandra Aldinucci,† Antonio Turco,‡ Tiziana Biagioli,† Francesca Maria Toma,‡ Daniele Bani,§ Daniele Guasti,§ Cinzia Manuelli,§ Lisa Rizzetto,†,‡,§,∥ Duccio Cavalieri,†,‡,§,∥ Luca Massacesi,† Tommaso Mello,⊥ Denis Scaini,□ Alberto Bianco,¶ Laura Ballerini,*,□ Maurizio Prato,*,‡ and Clara Ballerini*,† †
Department of NEUROFARBA, University of Florence, 50134 Firenze, Italy Department of Chemical and Pharmaceutical Sciences, University of Trieste, 34127 Trieste, Italy § Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy ∥ Research and Innovation Center, E. Mach Foundation, San Michele all’Adige, 38010 Trento, Italy ⊥ Department of Biomedical Sciences, University of Florence, Florence, Italy ¶ CNRS, Institut de Biologie Moléculaire et Cellulaire, Laboratoire d’Immunopathologie et Chimie Thérapeutiques, 67000 Strasbourg, France □ Life Science Department, University of Trieste, 34127 Trieste, Italy ‡
S Supporting Information *
ABSTRACT: Nanomaterials interact with cells and modify their function and biology. Manufacturing this ability can provide tissue-engineering scaffolds with nanostructures able to influence tissue growth and performance. Carbon nanotube compatibility with biomolecules motivated ongoing interest in the development of biosensors and devices including such materials. More recently, carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. To gather further knowledge on the true potential of future constructs, in particular to assess their immunemodulatory action, we evaluate carbon nanotubes interactions with human dendritic cells (DCs). DCs are professional antigen-presenting cells and their behavior can predict immune responses triggered by adhesion-dependent signaling. Here, we incorporate DC cultures to carbon nanotubes and we show by phenotype, microscopy, and transcriptional analysis that in vitro differentiated and activated DCs show when interfaced to carbon nanotubes a lower immunogenic profile. KEYWORDS: MWCNTs, DCs, cytoskeleton, gene expression
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self-or not-self-antigens to lymphocytes. Along with these processes, DCs vary phenotype and function, shifting from antigen processing cells to professional antigen-presenting ones.7 Recent reports8,9 showed that solid tissue-growth platforms modify DC adhesion properties and instruct different inflammatory responses by guiding differential DC maturation, phenotype expression, and production of pro- or antiinflammatory cytokines. These data suggest that the extent at which artificial scaffolds or implants modify the adhesion of
anotechnologies are increasingly used in tissue-engineering strategies to improve the interface between artificial scaffolds and cells1,2 and to investigate the ability of manufactured physical-chemical features to activate cell molecular machinery able to translate the emerging signaling into tissue specific instructions.3−6 Crucial to the exploitation of artificial scaffolds in organ repair is to investigate the immune system response to nanoscale three-dimensional architectures able to sustain tissue growth. Within the immune system, human myeloid dendritic cells (DCs) are key regulators of tolerance induction or, on the opposite, of innate and adaptive immune responses. DCs behave as circulating sentinels, continuously moving from nonlymphoid to lymphoid tissues, able to process and present © 2013 American Chemical Society
Received: September 11, 2013 Revised: November 5, 2013 Published: November 13, 2013 6098
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Figure 1. Evaluation of morphology, viability and differentiation state of DCs adhering to MWCNTs. (a) Time-lapse montage (10′ total time) showing dynamic spreading (green box) and detachment (red box) of DCs at day 5 of culture on MWCNTs. Full video available as Supporting Information Movie 1. (b) Percentage of adherent cells to MWCNTs with respect to the total cell number along the culture time. (c) SEM image shows a DC tightly attached to substrate with the cell dendrites that interdigitate with nanotubes. (d) At day 6 of culture, DCs were analyzed by flow cytometry for vitality by PI incorporation (upper panel) and for differentiation state from monocytes by the CD11c/CD14 expression (lower panel). One experiment representative of three independent ones is shown.
circulating DCs will interfere with the complex dynamic state of these cells and will tune immunogenic reactions, thus representing a supplementary strategy to regulate immune function while allowing tissue repair.10,11 Our approach to address this issue was to incorporate DC cultures to artificial conductive nanostructures, such as carbon nanotubes. Indeed, recently, carbon nanotubes have attracted tremendous attention for the development of nanobiohybrid systems able to govern cell-specific behaviors in cultured neuronal networks and explants12−21 and have been shown to promote viability and proliferation of neonatal cardiac myocytes.22 Carbon nanotubes are graphene sheets rolled into nanoscale dimension single- or multiwalled cylindrical tubes, SWCNTs and MWCNTs, respectively;23 both these nanotube geometries have been extensively studied in a wide range of bioapplications.24 In in vivo and in vitro experiments, MWCNTs have been shown to be blood compatible and to be a suitable scaffold for bone regeneration,25,26 cultured synaptic network formation,12−21 and neonatal cardiomyocite maturation.22 Being internalized by cells, functionalized carbon nanotubes are attractive as multifunctional cargo systems opening up new opportunities in chemical, biological, and medical applications.27 In addition, MWCNTs have been exploited as molecular carriers for gene delivery.28,29 Besides these exciting developments, monitoring MWCNTs in live tissues and cells, assessing cytotoxicity and evaluating their impact on inflammatory cascades and immune reaction is the fundament for their future applications.30−33
In the present work we investigate the interaction between MWCNT scaffolds and human myeloid DCs. Here we show that in vitro differentiated and activated DCs, when attached on carbon nanotube interfaces, do not activate cell death programs. More interestingly, DCs entangled to MWCNTs undergo a phenotypic and functional shift, displaying a lower immunogenic profile. To address the mechanistic pathways between the enhanced tolerogenic profile of DCs and their contact to MWCNT scaffolds, we perform functional studies associated to electron and confocal microscopy analysis of cell/substrate contacts, together with gene expression analysis. MWCNTs induce cell modifications by contacts and due to their geometrical and physicochemical properties ultimately modulate DC-mediated immune function. In the present work, MWCNTs were functionalized to make them highly dispersible in water (Supporting Information). The degree of functionalization was evaluated by thermal gravimetric analysis (TGA; Supporting Information Figure S1), the integrity and the increase in the dispersibility of the functionalized MWCNTs (Supporting Information Figure S1b) respect to the pristine MWCNTs (Supporting Information Figure S1a) was evaluated by transmission electron microscopy (TEM) analyses. A dispersion of functionalized MWCNTs ( f-MWCNTs) was drop casted on the glass substrate to obtain a thick carpet of MWCNTs and subsequently heated at 350 °C under nitrogen atmosphere (Supporting Information; see images of such a substrate in Figure 1c and in the inset of Figure 5). This procedure is sufficient to induce the defunctionalization of f-MWCNTs 6099
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Figure 2. Effects of MWCNTs on DC function and phenotype. DCs differentiated and activated with LPS in control conditions (CTR DCs) or in the presence of MWCNTs (MWCNT adhering DCs or MWCNT floating DCs; the latter, being consistently similar to control DCs, were not included in panel c) were examined for allostimulatory ability, phenotype, micropinocytosis capability, and cytokine profile. (a) DC allostimulatory ability was tested in a mixed lymphocyte reaction (MLR) with allogeneic CD4+ T cells; three different doses of DCs were used (x-axis). The T cell proliferative response (y-axis) is reported as mean value of counts per minute (CPM); one experiments representative of five independent ones is shown. (b) The expression of cytokines IL-23p19; IL-12p35; IL-6; IL-10; and TNFα was evaluated by real time PCR; relative expression of mRNA levels (y-axis) was determined by comparing experimental levels with a standard curve, using β-Actin as housekeeping gene for normalization. One experiment representative of three independent ones is reported. (c) Micropinocytosis ability was assessed by dextran-FITC uptake assay, performed before (upper histogram) and after LPS exposure (lower histogram); x-axis, Dextran-FITC fluorescence intensity; y-axis, number of acquired events; one experiment representative of three independent ones is shown. (d,e) The surface expression of a typical panel of DC maturation markers, HLADR, CD80, CD86, and CD83, was evaluated by flow cytometry in DCs cultured on MWCNTs and polystyrene culture plates (CTR) (d) and amorphous carbon (carbon), histogram (e). The percentage of positive cells for each staining is reported on x-axis (mean values ± SD of 5 different experiments). (e) (Top) AFM imaging of drop-casted amorphous carbon reveals submicrometrical globular features characterized by local nanopatterned roughness. (Bottom) Cells adhering on such substrates are shown at low magnification in bright-field microscopy.
preserving the structure of the nanotubes as observed by TEM and TGA (plots in Supporting Information Figure S1; see also ref 19). In order to address whether MWCNTs affected the in vitro maturation of DCs from CD14+ monocytes progenitors, monocytes-derived DCs (density 1 × 106 cell/ml) were cultured and grown for 6 days (medium containing GMCSF and IL-4; Supporting Information) in control conditions (standard culturing plates) or in the presence of MWCNT substrates. In control samples, during the first 4 days of culturing, adherent cells detached upon minimal mechanical manipulation, preventing to distinguish between adherent and suspended cells in any further analysis. In fact, in control after 4 days of culturing virtually all DCs were detected as floating cells in suspension.34 In the presence of MWCNTs, the fraction of cells adherent to the nanostructured substrate displayed a higher mechanical stability; this allowed the separation and characterization of the two different populations, adherent and floating DCs, along with time in culture. We found that the percentage of adherent cells in the presence of MWCNT with respect to the total cell number decreased along with the culture time from 50% at day 1 to 3% at the end of culture (day 6; Figure 1b). We hypothesize that during
culturing, cells may attach and detach from MWCNTs several times with only a progressively smaller fraction remaining stably adherent. We further explore DC behavior in real time identifying by time-lapse video microscopy (Figure 1a for MWCNT cultures; Supporting Information Movie 1), the adherent DCs fraction from those floating in suspension along with their in vitro development and the dynamic of DC adhesion and detachment from the substrate (Figure 1a and Supporting Information Movie 1). The time-lapse video supports our hypothesis of a dynamic interplay between DCs attachment and detachment on and from MWCNTs. We decided to investigate whether adhesion to carbon nanotubes transiently affected DCs maturation and responses when compared to floating cells. Given the dynamic of attachment and detachment, we decided to compare adherent DCs toward floating DCs in MWCNT samples and we used in each experimental series the floating DCs of controls as a benchmark of the features of these cells when not exposed to MWCNTs. The fraction of adherent DCs, although small in culturing conditions, becomes relevant in the perspective of predicting certain immune responses in vivo toward nanostructured interfaces. Therefore, in the presence of MWCNTs we target the stably adherent fraction of DCs, virtually absent in controls 6100
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Figure 3. Effect of MWCNTs on DC cytoskeleton organization. DC actin distribution upon LPS activation was analyzed by confocal microscopy (60× magnification). Upper panel, control DCs; lower panel, DCs adhering to MWCNTs. Green, f-actin; red, CD11c. Colocalization is evidenced by xz view. One experiment representative of three independent ones is shown.
amorphous carbon-coated substrates (representing a nanostructured control for the MWCNT substrate, Figure 2e atomic force microscopy (AFM) panel and see Supporting Information). We further tested DC ability to express polarizing and proinflammatory cytokines in response to LPS stimulation. MWCNT-adherent DCs were not able to produce detectable amounts of transcripts for IL-12p35 and IL-23p19 coding genes when compared to MWCNT-floating and controls, where the relative β-actin expression was 2.3 and 3.4, respectively (Figure 2b). The presence of MWCNTs strongly reduced relative expression of IL6 and IL10 genes; on the contrary, adhesion to MWCNTs quite enhanced TNFA expression. Taking into account these results, it is tempting to hypothesize that the adhesion to MWCNTs mechanically instructs DCs, specifically affecting the production of cytokines that need DC cytoskeleton polarization to be produced, while leaving unperturbed those cytokines homogeneously produced in the cytoplasm. This is also supported by the observed effect of MWCNTs on the micropinocytosis, a DC feature typically regulated by adhesion and cytoskeleton organization. We evaluated micropinocytosis by dextran-FITC uptake assay (Supporting Information) performed before and after LPS exposure (Figure 2c). In the absence of stimulation, control cells have the maximum value (97%) of dextran-FITC uptake, while MWCNT adherent DCs resulted poorly efficient in micropinocytosis, as demonstrated by the low (39%) dextranFITC positive cell percentage in our experiments (Figure 2c). The LPS stimulation minimizes the differences between the three cell populations, since control and suspension DCs reduced, as expected, their endocytosis ability whereas MWCNT-adherent DCs maintained the same behavior shown before LPS treatment. Therefore, we suggest that
that remained tightly attached to the substrate with the cell dendrites interdigitated with the carbon nanotube structures (Figure 1c; scanning electron microscopy, SEM). We investigated, comparing control- and MWCNT-floating versus MWCNT-adherent DCs, how the substrate affected both vitality and differentiation of precursor CD14+ cells. At day 6, differentiated DCs were first analyzed by flow cytometry for vitality by propidium iodide (PI) incorporation and for phenotype (Figure 1d). Control DCs and those cultured in the presence of MWCNTs, adherent or not, were viable and fully differentiated, as shown by the low amount (0.2−0.3%) of PI positive cells together with the loss (around 0.2%) of monocyte marker CD14 concomitant with the presence (99.5%) of CD11c. To investigate how the adhesion to MWCNTs affected cell function, DCs were differentiated and activated with LPS (Supporting Information). DC allostimulatory ability was tested in a mixed lymphocyte reaction (MLR) with allogeneic CD4+ T cells (Figure 2a). MWCNT-adherent DCs were significantly less efficient in inducing T cell proliferation with respect to MWCNT-floating DCs and to controls (p < 0.0002 at the dose of 10000 DCs/well; p < 0.004 at the dose of 1000 DCs/well). We strengthened this functional observation by further checking the cell phenotype upon LPS stimulation. In fact, the expression of the typical DC maturation marker, CD80, was dramatically deficient in DCs attached to MWCNTs, when compared to controls and to MWCNTfloating DC population (p = 0.0002) (Figure 2d). In the same set of experiments, HLA-DR, CD86, and CD83 were unaltered within the three cell populations (control-, MWCNT-floating, and MWCNT-adherent). To stress the specificity of adhesion to MWCNTs in inducing a change in phenotype, Figure 2e shows results obtained from DCs grown in the presence of 6101
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This overview indicated that MWCNTs affect DC cellular functions, such as their ability to adhere (focal adhesion, FAs, and adherent junction, AJs, organization) and their ability to properly reorganize the cytoskeleton upon LPS recognition (Figure 4 and Supporting Information Figure S2). We observed that carbon nanotubes negatively influences PI3K signaling and the pathways regulated by it (Figure 4 and Supporting Information Figure S2). Furthermore, the presence of significantly down-regulated, or not-affected, immune response pathway strengthened our observation that MWCNTs altered the ability of DCs to respond to LPS. Notably, the coding gene of sphinophilin, a well-known PDZ domain protein that serves as scaffold in the construction of neuronal synapses36 and more recently was shown to be equally recruited in immunological synapses,37 in our data is down regulated (PPP1R9B, log2 FC = −2.66). We also observed over-representation of pathways and categories related to eicosanoids, arachidonic, and cholesterol content regulation, suggesting a changing in cell lipid composition (Table 1, Supporting Information Figure S2). The affected pathways we identified are consistent with the observed physiological behavior: MWCNT-adherent DCs do not properly rearrange cytoskeleton and this affects endocytic processes as well as fully activation ones. This observation is supported by the reduced PI3K/AKT signaling, due to upregulation of PTEN gene in combination with a downregulation of PI3K and AKT1 genes (log2 FC = 1.101, −2.161, and −1.519, respectively; Figure 4 and Supporting Information List 1). A similar gene regulation was previously observed in DCs from aged people, which showed a reduced capacity to phagocytosis through micropinocytosis and endocytosis.38 The overexpression of FA pathways related genes in particular the masterregulator BCL2 and the overexpression of PTEN and HMOX, crucial genes in cell adhesion, are in agreement with the overexpression of cell cycle related genes. This observation, strengthened by the functional and phenotypic data, suggests the presence of immature and/or less immunogenic DCs. It is interesting to note that DCs maturation program is known to be associated with cell commitment. Altogether, our data further support the ability of a substrate in modifying cell contact surface and attachment, thus modulating cell functions via an alteration in cytoskeleton organization and in cell functional polarization. Scattered DCs were found adherent to the underlying nanotube-coated glass surface. These cells showed typical ultrastructural characteristics of activated monocyte-derived antigen-presenting cells, such as numerous cell processes and electron-dense lysosomes in the cytoplasm. These cells were often observed in close contact with the nanotube substrate, which in some cases were surrounded by dendrites and resulted after microtome cuts as incorporated structures (Figure 5, TEM image, SEM inset, and cartoon). We may conclude that DCs sense the substrate and react changing structure organization. This may occur through an integration of mechanical and biochemical signals that lead to less immunogenic cell phenotypes. We propose that a genuine adhesion-mediated mechanism is involved in the modulation of DC function when sensing MWCNT scaffolds. This finding provides a deeper understanding of DC behavior while strengthening the general role of adhesion-mediated interactions with implantable nanostructures. Cells can act as mechanosensitive units responding to the mechanical stimulation of the extracellular matrix through focal adhesions and via changes in their cytoskeletal organiza-
MWCNTs affect DC polarization and related activation, without interfering with cell maturation processes. Because both micropinocytosis and polarization processes are mediated by cytoskeleton rearrangements, we investigated the influence of MWCNTs in the DC actin distribution. The presence of such changes may link the cytoskeletal features to the functional/phenotypic changes observed. As shown by confocal microscopy (Figure 3, upper panels), upon LPS activation in control DCs the f-actin was distributed along the cell surface, mainly concentrated at the level of dendrites, and did not colocalize with CD11c, as expected for fully mature/ polarized DCs. On the contrary, in MWCNTs adherent DCs in differentiated and activated cells f-actin and CD11c displayed an identical distribution and colocalized (Figure 3, lower panels). These data strongly support the idea that carbon nanotube scaffolds, probably via adhesion-mediated mechanisms, affect the ability of DCs to polarize, which is a crucial prerequisite for functional immune synapse (IS) formation. To investigate the effects of carbon nanotubes on DC reprogramming, we analyzed and compared the transcriptional response of MWCNT-adherent DCs with that of MWCNTfloating ones. Because we observed that adherent DCs displayed an altered costimulatory molecules expression and endocytosis process, we performed the transcriptional analysis upon 4 h of stimulation with LPS, in order to address whether during the differentiation program MWCNTs affect DC maturation and presentation properties. Differentially expressed genes (DEGs) analysis identified a total of 650 down-regulated and 305 up-regulated genes (Supporting Information List 1). To investigate the functional properties of genes whose expression was changed in adherent DCs, we performed gene ontology enrichment over biological process. Starting from this, we identified categories in which gene-expression resulted differently modulated by MWCNTs. Among the identified genes, several are involved in the regulation of actin cytoskeleton, adhesion properties, cell communication, and integrin signaling as well as Rho GTPase-mediated transduction signaling (Table 1). To investigate the regulation of pathways and cellular networks in our samples, we performed a pathway analysis using pathway signatures35 (Supporting Information Figure S2). Table 1. Selection of the Most Significant Enriched Gene Ontology Biological Processes upon down and up Differentially Expressed Genes Down-Represented GO Biological Process corrected p value
category membrane invagination endocytosis membrane organization and biogenesis immune response small GTPase mediated signal transduction actin cytoskeleton organization and biogenesis regulation of small GTPase mediated signal transduction Up-Represented GO Biological Process category response to oxidative stress sterol metabolic process isoprenoid metabolic process response to reactive oxygen species cellular lipid metabolic process
1.21 1.21 1.21 1.50 7.47 2.59 3.03
× × × × × × ×
10−04 10−04 10−04 10−03 10−03 10−02 10−02
corrected p value 2.38 3.70 4.29 5.33 3.02
× × × × ×
10−03 10−03 10−03 10−03 10−02 6102
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Figure 4. Affected genes by MWCNT substrate modulation of DCs properties. Transcriptional analysis was performed on DCs cultured for 4 h in presence or absence of LPS, after differentiation in the presence of carbon nanotubes. After pathway analysis, affected genes were calculated from the standard deviation from the median on all the genes. Genes that also resulted as differentially expressed (DEGs) applying a locally weighed linear regression (LOWESS) analysis are represented in white.
mediated by cell−cell contacts,42 when DCs do not undergo to the molecular and cytoskeletal organization known as IS. We show here that DC function is modulated by contact with the synthetic nanosubstrate in particular DCs sensing MWCNT platforms become less immunogenic. We suggest that this effect is, at least in part, due to a loss of polarization ability, a mechanism that has been recently described.43 In fact, upon TLR stimulation DCs undergo a reorganization of the cytoskeleton at the IS; such a reorganization polarizes the production of cytokines and vesicles, via Cdc42 expression, ultimately affecting the fate of stimulated T cells. To support this hypothesis in our transcriptional data we report a down regulation of Cdc42 effector protein 2 that may act downstream Cdc42 and of Cdc42 small effector 1 (log2 FC = −1.151, Supporting Information List 1), potentially involved in cell shape changes and phagocytic events in macrophages. This result is consistent with the detected reduction in expression of IL-12 and IL-23 genes, leaving the whole cytokine expression machinery unperturbed. We may speculate that a similar process involves the differential expression of costimulatory molecules with CD80 more susceptible to the modulation brought about by cytoskeleton reorganization. The ability of MWCNTs to effect DC actin cytoskeleton may thus be mediated by contacts and adhesion. Further support to this interpretation is provided by our recent work42 where we reported that immune responses and IS modulation during cell−cell contact interactions required actin cytoskeleton reorganization to change cell function. The SEM and TEM analysis document DCs tight interactions with the MWCNT carpets with DC processes establishing close contacts with the
Figure 5. Ultrastructural features of DCs grown on MWCNT layers. Representative transmission electron micrograph of a DC cultured for 6 days on carbon nanotubes. The cell established close contacts with the surrounding nanotubes (arrows and high-power insets), one of which (right) is included into a plasma membrane in-folding located in their peripheral cytoplasm. This DC appears viable with no signs of damage. The right panel depicts the presumptive 3D relationship between the cells and MWCNT carpet. A detail on the MWCNT carpet surface is depicted in the SEM inset (bottom right).
tion.39,40 The specificity of MWCNTs in promoting adhesionmediated regulation of the immunogenic profile of DCs is strengthened by previous results which documented how DCs adhesion to various substrates (including poly-L-lysine) variably induced different DC immunogenic profiles,8,41 therefore the mere fact that DCs are attached to a substrate is not per se predictive of immunogenic reaction. Furthermore, our interest is on MWCNTs and on the possibility that carbon nanotube nanofeatures mimic what has been previously shown to be 6103
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Author Contributions
nanomaterial and transcription data confirm that cytoskeleton rearrangement may be at the origin of the observed cell phenotype. Differently from van Den Dries9 findings, our 3D surface does not boost DCs toward a more immunogenic phenotype due to podosome relocation; probably the different scale of substrate or its composition is responsible of the different effect.44 Finally, there is a certain debate on toxicity of carbon nanotubes in their soluble form27,45,46 and this is dependent, in cells as macrophages, on material internalization with consequent inflammatory response and cell apoptosis.47 In the present work, we show that MWCNTs do not induce cell death. First, we show it in flow cytometry PI experiments and second in mRNA microarray analysis. Nonetheless, potential carbon nanotube toxicity remains a concern, particularly in mass-scale, industrial applications.27 However, it is emerging that carbon nanotubes geometry and surface chemical functionalization diminishes or altogether abolishes the inflammatory response and granuloma formation associated with this material, thus strongly influencing carbon nanotube biocompatibility.48−50 Future acceptance of carbon nanotube based technology in medicine requires a deeper understanding of immune responses to these materials. In our experiments DCs cultured on MWCNTs, once detached are further cultivated in a MLR reaction that last 6 days and their vitality is not affected. Furthermore, from live microscopy observations apparently cell vitality is not varying during this period. Therefore, we can exclude a more subtle toxic effect of MWCNTs with a progressive switching off of adhesion related genes. Ultimately, DCs are cells that rapidly react to contact, regardless whether it is a cell−cell contact or a substrate−cell contact and this behavior probably contributes to their ability to differently act in different tissues, that is, derma or lymphonodes. Altogether these results suggest that altering topographical and physical features of growth surfaces holds the potential to tune immune reaction modulation, mediated by migrating DCs being able to attach more than once to the interfaces with a focus on cell polarity alteration. We may therefore hypothesize that different substrates are able to instruct cell responses by different mechanisms that involve cytoskeleton modification with consequent alteration of immune function. MWCNTs can be incorporated into constructs to produce neural interfaces with high interfacial areas, conductivity, and electrochemical stability.51,52 In this framework, investigating in vitro differentiated and activated DCs phenotypic/functional shift when interfaced to MWCNTS holds the potential to predict the immune response toward electrodes and provide a valuable tool to tackle a key challenge in engineering neural implants.
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A.A. and T.B. design and performed all cell biology, confocal, and RT-PCR experiments; T.M. performed time-lapse videomicroscopy; A.T. and F.M.T. purified, analyzed, and prepared the MWCNT substrates and provide SEM, TEM,and TGA experiments on MWCNT; D.B. and D.G. design and performed the TEM experiments on biological specimens; L.R., DC., and C.M. designed and performed the transcriptional and pathways expression analysis; L.M. provided funding; D.S. performed AFM measures; A.B. contributed to the experimental design; L.B., M.P., and C.B. conceived the study, contributed to the experimental design and analysis of data, and provided funding; C.B. and L.B. wrote the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are especially grateful to E. Traggiai and H. Dumortier for their helpful comments and for their critical revision of the manuscript. Financial support from ERC grant CARBONANOBRIDGE No. 227135 and NEUROSCAFFOLDS No. 604263 is gratefully acknowledged.
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ABBREVIATIONS AJ: adherent junctions; AFM: atomic force microscopy; DCs: dendritic cells; DEG: differentially expressed genes; FA: focal adhesion; GO: gene ontology; IS: immune synapse; LPS: lipopolysaccharide; MLR: mixed lymphocyte reaction; MWCNTs: multiwalled carbon nanotubes; SEM: scanning electron microscopy; TEM: transmission electron microscopy; TLR: Toll-like receptors
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(1) Place, E. S.; Evans, N. D.; Stevens, M. M. Complexity in Biomaterials for Tissue Engineering. Nat. Mater. 2009, 8, 457−470. (2) Dvir, T.; Timko, B. P.; Kohane, D. S.; Langer, R. Nanotechnological Strategies for Engineering Complex Tissues. Nat. Nanotechnol 2011, 6, 13−22. (3) Discher, B. M.; et al. Design of Amphiphilic Protein Maquettes: Controlling Assembly, Membrane Insertion, and Cofactor Interactions. Biochemistry 2005, 44, 12329−12343. (4) Cruz, L. J.; et al. Multimodal Imaging of Nanovaccine Carriers Targeted to Human Dendritic Cells. Mol. Pharmaceutics 2011, 8, 520− 531. (5) Kou, P. M.; Schwartz, Z.; Boyan, B. D.; Babensee, J. E. Dendritic Cell Responses to Surface Properties of Clinical Titanium Surfaces. Acta Biomater. 2011, 7, 1354−1363. (6) Shankar, S. P.; Petrie, T. A.; Garcıa, A. J.; Babensee, J. E. Dendritic cell responses to self-assembled monolayers of defined chemistries. J. Biomed. Mater. Res. A 2010, 92, 1487−1499. (7) Liu, K.; et al. In vivo analysis of dendritic cell development and homeostasis. Science 2009, 324, 392−397. (8) Acharya, A. P.; Dolgova, N. V.; Clare-Salzler, M. J.; Keselowsky, B. G. Adhesive substrate-modulation of adaptive immune responses. Biomaterials 2008, 29, 4736−4750. (9) van den Dries, K.; et al. Geometry sensing by dendritic cells dictates spatial organization and PGE2-induced dissolution of podosomes. Cell. Mol. Life Sci. 2012, 69, 1889−1901. (10) Contento, R. L.; Campello, S.; Trovato, A. E.; Magrini, E.; Anselmi, F.; Viola, A. Adhesion shapes T cells for prompt and sustained T-cell receptor signalling. EMBO J. 2010, 29, 4035−4047. (11) Berzat, A.; Hall, A. Cellular responses to extracellular guidance cues. EMBO J. 2010, 29, 2734−2745.
ASSOCIATED CONTENT
S Supporting Information *
Detailed materials and methods, Figures S1 and S2, List 1, Movie 1, figure legends, and Movie 1 legend. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*(C.B) E-mail: clara.ballerini@unifi.it Phone:+390554271377. Fax: +39 0554271380. *(M.P) E-mail:
[email protected]. *(L.B) E-mail:
[email protected]. 6104
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Nano Letters
Letter
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