Macrophage sensing of single-walled carbon nanotubes via Toll-like receptors

1 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 www.nature.com/scientificreports Macrophage sensing of singlewalled carbon nanotubes via Tolllike receptors Sourav P. Mukherjee1, Olesja Bondarenko1,2, Pekka Kohonen 1, Fernando T. Andón1,3, Táňa Brzicová1,4, Isabel Gessner5, Sanjay Mathur5, Massimo Bottini6,7, Paolo Calligari8, Lorenzo Stella 8, Elena Kisin9, Anna Shvedova9,10, Reija Autio11, Heli Salminen-Mankonen12, Riitta Lahesmaa12 & Bengt Fadeel1 Carbon-based nanomaterials including carbon nanotubes (CNTs) have been shown to trigger inflammation. However, how these materials are ‘sensed’ by immune cells is not known. Here we compared the effects of two carbon-based nanomaterials, single-walled CNTs (SWCNTs) and graphene oxide (GO), on primary human monocyte-derived macrophages. Genome-wide transcriptomics assessment was performed at sub-cytotoxic doses. Pathway analysis of the microarray data revealed pronounced effects on chemokine-encoding genes in macrophages exposed to SWCNTs, but not in response to GO, and these results were validated by multiplex array-based cytokine and chemokine profiling. Conditioned medium from SWCNT-exposed cells acted as a chemoattractant for dendritic cells. Chemokine secretion was reduced upon inhibition of NF-κB, as predicted by upstream regulator analysis of the transcriptomics data, and Toll-like receptors (TLRs) and their adaptor molecule, MyD88 were shown to be important for CCL5 secretion. Moreover, a specific role for TLR2/4 was confirmed by using reporter cell lines. Computational studies to elucidate how SWCNTs may interact with TLR4 in the absence of a protein corona suggested that binding is guided mainly by hydrophobic interactions. Taken together, these results imply that CNTs may be ‘sensed’ as pathogens by immune cells. Carbon-based nanomaterials including carbon nanotubes (CNTs) and graphene oxide (GO) are potential candidates for various applications in medicine such as drug delivery and imaging1. However, the successful translation of nanomaterials for biomedical applications requires a detailed understanding of the biological interactions of the materials. In particular, interactions of nanomaterials with the immune system, the first line of defense against foreign intrusion, are of key importance2. The innate immune system is deployed in defense against microorganisms and involves the recognition of pathogen-associated molecular patterns (PAMPs) by (PRRs) on the surface of phagocytic cells. The immune system also responds to tissue damage, a process that is triggered by so-called danger or damage-associated molecular patterns (DAMPs)2. We previously hypothesized that nanoparticles might be recognized directly as nanoparticle-associated molecular patterns or NAMPs by cells of the immune system3. However, experimental evidence to support this idea was largely lacking. Hence, while there 1Nanosafety & Nanomedicine Laboratory, Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, 17177, Stockholm, Sweden. 2Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Tallinn, 12618, Estonia. 3Laboratory of Cellular Immunology, Humanitas Clinical and Research Institute, 20089, Rozzano-Milano, Italy. 4Department of Genetic Toxicology and Nanotoxicology, Institute of Experimental Medicine AS CR, 14220, Prague, Czech Republic. 5Inorganic and Materials Chemistry, University of Cologne, 50939, Cologne, Germany. 6Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, 00173, Italy. 7Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, 92037, USA. 8Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Rome, 00133, Italy. 9Exposure Assessment Branch, National Institute for Occupational Safety and Health, Morgantown, WV, 26505, USA. 10Department Pharmacology & Physiology, West Virginia University, Morgantown, WV, 26505, USA. 11Faculty of Social Sciences, University of Tampere, 33014, Tampere, Finland. 12Turku Centre for Biotechnology, University of Turku, 20520 Turku, and Åbo Akademi University, 20500, Turku, Finland. Sourav P. Mukherjee and Olesja Bondarenko contributed equally to this work. Correspondence and requests for materials should be addressed to B.F. (email: [email protected]) Received: 10 May 2017 Accepted: 3 January 2018 Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 is an emerging body of literature on nanomaterial effects on the immune system, there are few if any studies in which evidence for specific ‘sensing’ of nanoparticles by immune-competent cells has been provided. Previous work has shown that proteins adsorbed onto the surface of nanoparticles can activate macrophages via surface receptors, resulting in the secretion of pro-inflammatory cytokines4, but whether nanomaterials themselves are recognized by immune cells through specific receptors is not known. Single-walled and multi-walled CNTs as well as carbon nanofibers have been shown to induce pro-inflammatory and pro-fibrotic responses, especially following pulmonary exposure5. Specifically, exposure to SWCNTs was shown to elicit acute inflammation and early-onset fibrosis in the lungs of mice, with neutrophil accumulation, followed by macrophage influx, and an early elevation of pro-inflammatory cytokines followed by production of pro-fibrotic cytokines6. Moreover, elevated numbers of dendritic cells (DCs) are found in the lungs of mice following pharyngeal aspiration of SWCNTs7. Furthermore, based on the literature available at the time, certain rigid and ‘needle-like’ MWCNTs were classified by the International Agency for Research on Cancer (IARC) as being potentially carcinogenic to humans8, and a more recent, in-depth examination of in vivo and in vitro studies has affirmed the original evaluation that some MWCNTs are potentially carcinogenic, while the data are inconclusive for others9. For graphene-based materials, a consensus on toxicity or health risks has yet to emerge, although considerable efforts are being invested in order to address this question in a systematic fashion10,11. In a recent study, so-called graphene nanoplatelets were shown to induce pulmonary toxicity in mice at high doses, but no lung fibrosis12. In another recent inhalation study in rats, graphene nanoplatelets showed low toxicity, with no distinct pathology or inflammation; the materials were ingested by lung macrophages13. In a study on single-layer GO sheets with lateral dimensions below 500 nm, no significant cytotoxic responses were noted using A549 lung carcinoma cells, and no inflammation or granuloma formation was observed in vivo following intraperitoneal injection14, while a more recent study suggested that the impact of GO on human peripheral blood-derived cells was dependent on the lateral dimensions of GO15. Here we present detailed mechanistic in vitro studies to address the impact of well-characterized and endotoxin-free SWCNTs and GO on primary human macrophages. Guided by global transcriptomics analysis of macrophages exposed to these materials, we focused our studies on chemokine signaling and could show that SWCNTs, but not GO, induced chemokine secretion in macrophages. Our experimental results suggested that SWCNTs were sensed by Toll-like receptors (TLRs), PRRs on the surface of phagocytic cells16. Cellular uptake of SWCNTs was not required for chemokine signaling in exposed macrophages. Molecular docking suggested that SWCNTs may interact with TLR4 both via the tip and the side-walls. These studies indicate that immune cells are able to ‘sense’ SWCNTs through specific immune receptors and as such are relevant for our understanding of the impact of these materials on human health. Results Characterization of carbon-based nanomaterials. SWCNTs, produced by the high pressure CO disproportionation process (HiPco) technique, and GO, synthesized by a modified Hummer’s method, were characterized using an array of analytical techniques. Transmission electron microscopy (TEM) revealed that SWCNTs had an average diameter of 1–4 nm and an average length of 0.5–2 µm, whereas GO had an average diameter (lateral size) of 1.1 ± 0.3 µm. The surface charge (ζ-potential) was −42.3 ± 0.9 mV and −42.0 ± 1.2 mV for SWCNTs and GO, respectively. The samples were also characterized following dispersion in cell culture medium (i.e., DMEM) with and without 10% FBS (Fig.S3a–e). The ζ-potential remained negative in DMEM + FBS, though less negatively charged when compared to samples dispersed in water. DLS measurements suggested that SWCNT and GO were less agglomerated in DMEM + FBS than in medium without FBS, though DLS results for non-spherical objects should be interpreted with caution. Nanomaterials are frequently contaminated with lipopolysaccharide (LPS) or endotoxin, the cell wall component of Gram-negative bacteria17. Therefore, both materials were tested by using the conventional limulus amoebocyte lysate (LAL) assay and found to be endotoxin-free (data not shown). Moreover, in order to exclude potential artefacts due to interference with the assay, which could skew the interpretation of the biological data, we also performed a macrophage activation test based on the evaluation of TNF-α secretion by primary human monocyte-derived macrophages (HMDM) in the presence and absence of a specific LPS inhibitor18. LPS (100 ng/ mL) was included as a positive control. Using this approach, the CNTs were confirmed to be endotoxin-free, as macrophage secretion of TNF-α in response to CNTs was very low and not affected by polymyxin B (Fig.S1a–c). Cytotoxicity assessment and cellular uptake. We then assessed both nanomaterials for cytotoxicity using the lactate dehydrogenase (LDH) release assay. ZnO nanoparticles (100 µg/mL) which are known to be cytotoxic for macrophages19, were included as a positive control. SWCNT exposure resulted in a slight, dose-dependent increase in LDH release at 24 h (14–16% more LDH release compared to the control at concentrations 30 and 100 µg/mL, respectively; p < 0.05), whereas GO did not yield any cytotoxicity in HMDMs (Fig.S1d). Next, we assessed for cellular uptake of the nanomaterials. TEM imaging confirmed that both nanomaterials were internalized by HMDM at 24 h (Fig.S2). SWCNTs and GO were mostly found within membrane enclosed vacuoles, suggesting that the uptake had occurred through an active, most likely endocytic process. Engulfment of GO changed its appearance from a 2D structure to a densely packed structure (Fig.S2). Microarray analysis of macrophage responses. To further evaluate the impact of the two nanomaterials on macrophages, we performed transcriptomics analyses following exposure for 6 h or 24 h to 10 or 30 µg/ml (i.e., sub-cytotoxic doses) of SWCNT or GO. Affymetrix® GeneChip® Human Genome U219 arrays were employed for global assessment of gene expression. The microarray data have been submitted to the Gene Expression Omnibus Database (GEO accession no. GSE83516). Few differentially expressed genes were noted at 6 h (data not shown) and we therefore focused our analysis on the 24 h exposure time-point. The nanomaterial exposure at the www.nature.com/scientificreports/ 3 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 latter time-point resulted in a specific transcriptional response with 52 differentially expressed genes in response to SWCNTs, while 7 genes were differentially expressed in response to GO (multigroup analysis by two-way ANOVA, p < 0.05, > 1.5-fold change compared to controls) (Fig.1a, and Supplementary Table1). Interestingly, the differentially expressed genes showed no overlap between the two nanomaterials. Subsequent canonical pathway enrichment analysis of differentially expressed genes using the Molecular Signatures Database showed that the three biological pathways with the most significant enrichment were those involved in cytokine-cytokine receptor interaction, chemokine signaling pathway and chemokine receptor binding pathway (multiple testing corrected p-values lower than 10−25) (Fig.1b, and Supplementary Table2). In addition, our analysis showed enrichment of the NF-κB pathway (comprising 7 genes, q-value 10−5.7). Indeed, analysis of transcriptional regulation networks showed that NF-κB was a central network connecting upregulated chemokines, and, moreover, that several members and targets of the NF-κB pathway were significantly modulated in response to SWCNTs (Fig.2a). Furthermore, upstream regulator analysis using the Ingenuity Pathway Analysis (IPA) software showed that the most significantly modulated NF-κB pathway network members were RELA (p65), IRF7 and NFKBIA (IκBα) (Z-scores 3.4, 2.9 and 2.4, respectively) (Fig.2b). Notably, according to a recent bioinformatics study, RELA and NFKBIA are both among the five key genes mediating NF-κB pathway-related inflammatory responses and macrophage activation20. Taken together, the transcriptomics analysis suggested that chemokine signaling pathways are prominently deregulated by SWCNTs, but not by GO, and that NF-κB is a potential upstream regulator of the transcriptional responses to SWCNTs. Validating the transcriptomics results. To validate these results, we assessed for macrophage production of chemokines. To this end, a multiplexed immunoassay for the detection of a defined set of cytokines/ chemokines following exposure to SWCNTs or GO (10–100 µg/mL) was applied. We focused the analysis on four chemokines (CCL3/MIP-1α, CCL5/RANTES, CXCL9 and CXCL10) based on the significant upregulation of the corresponding genes according to our transcriptomics analysis (above). As shown in Fig.3a–d, SWCNT-exposed cells produced high levels of all four chemokines, while GO-exposed cells failed to secrete these chemokines, which is thus in accordance with the microarray results. The secretion of CCL3 and CCL5 in SWCNT-exposed HMDM was dose-dependent (Fig.3e–h). These data thus corroborated the transcriptomics results. To further control for any potential endotoxin contamination, the CNTs were subjected to calcination at 250 °C to remove Figure 1. Transcriptomics analysis of macrophages exposed to carbon-based nanomaterials. Global gene expression profiling of human monocyte-derived macrophages (HMDM) was conducted after 24 h exposure to single-walled carbon nanotubes (SWCNTs) or graphene oxide (GO) (see Supplementary Table1). (a) Heatmap of differently expressed genes in response to 10 and 30 µg/mL GO (line 1 and 2, respectively) or 10 and 30 µg/ mL SWCNTs (line 3 and 4, respectively). Upregulated transcripts are presented in red and downregulated transcripts in green (log2 fold change > 0.75). The genes affected upon exposure to SWCNTs (n = 52) and GO (n = 7) did not overlap. (b) Canonical pathways modulated in HMDM after exposure to SWCNTs (see Supplementary Table2); ranking was performed according to multiple testing corrected p-value. www.nature.com/scientificreports/ 4 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 any residual endotoxins. The samples were recharacterized (in cell culture medium) and shown to display similar ζ-potential values while DLS measurements showed no significant changes (i.e., no agglomeration) (Fig.S4a,b). We then monitored the production of CCL5 in macrophages following exposure for 12 h to calcined SWCNTs (30 µg/mL) and found that the SWCNTs were still capable of triggering CCL5; moreover, this was not affected by polymyxin B, indicating that the observed effect is intrinsic to the SWCNTs and not a result of microbial contamination (Fig.S5a). Dissecting the signaling pathway. As noted above, upstream regulator analysis of the microarray data indicated that the NF-κB signaling pathway was involved in the transcriptional regulation of chemokine expression by SWCNTs. To validate this in silico prediction, HMDM were pretreated with the NF-κB inhibitor, Bay 11-7082, and chemokine secretion in response to SWCNT exposure was determined. SWCNT-induced production of CCL3 (Fig.4a) was significantly reduced upon preincubation with Bay 11-7082 (10 µM) and the secretion of CCL5 was completely blocked by the inhibitor (Fig.4b), thus confirming a role for NF-κB. Next we aimed to address how NF-κB is activated by SWCNTs. Figure 2. Upstream regulator analysis of the transcriptomics results. (a) The NF-κB network was identified as a potential upstream regulator of SWCNT-triggered responses in HMDM according to upstream regulator analysis (p < 0.01; Z-score > 2 S.D.). (b) Upstream regulator analysis73 of the data identified the modulation of NF-κB network members in HMDM exposed to SWCNTs for 24 h. Data were analyzed through the use of IPA (QIAGEN Inc., www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis). www.nature.com/scientificreports/ 5 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 Figure 3. SWCNTs, but not GO, trigger macrophage secretion of chemokines. Secretion of chemokines by primary human macrophages (HMDM) after a 24 h exposure to SWCNTs or GO as measured by a multi-plex immunoassay. (a–d) Exposure of HMDM to 30 µg/mL SWCNTs showed a significant increase in CXCL9, CXCL10, CCL3/MIP-1α, and CCL5/RANTES, while there was no response in cells exposed to GO at the same concentration. (e,g) Dose-dependent secretion of CCL3/MIP-1α, and CCL5/RANTES in cells exposed to SWCNTs, while there was no response to GO at any of the concentrations tested (f,h). Data are shown as mean values ± S.D. of three independent experiments using cells from different donors; p-values by Student’s t-test, * < 0.05; *** < 0.001. www.nature.com/scientificreports/ 6 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 The recognition of so-called PAMPs by different families of evolutionarily conserved PRRs (PRRs) initiates a signaling cascade that leads to the transcription of inflammatory cytokines and chemokines to eliminate pathogens and attract other immune cells to the site of infection21. In particular, Toll-like receptors (TLRs) play a key role in innate immunity16. TLRs activate multiple signaling pathways by recruiting adaptor proteins, such as myeloid differentiation factor 88 (MyD88), which initiate signal transduction pathways that culminate in the activation of transcription factors, eg., NF-κB, with ensuing cytokine/chemokine production. TLR4 is a receptor for bacterial LPS while TLR2 has specificity for multiple microbial components derived from bacteria, fungi, viruses, and mycoplasma21. The oxidized phospholipid, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) is known to inhibit LPS signaling via TLR2 and TLR422. To assess whether SWCNTs are capable of activating NF-κB via TLRs, we preincubated HMDM with oxPAPC (30 or 60 µg/mL) prior to exposure to SWCNTs (30 µg/mL). LPS (100 ng/mL) was included as a positive control. As shown in Fig.5a, oxPAPC significantly reduced LPS-induced secretion of CCL5. Moreover, SWCNT-induced production of CCL5 was completely blocked. Next, we tested whether inhibition of the adaptor protein, MyD88 would suppress NF-κB activation. To this end, cells were preincubated with Pepinh-MYD, a 26 aa peptide that blocks MyD88 signaling by inhibiting its homodimerization23. Pepinh-Control, a control peptide, was included as a negative control. Pepinh-MYD (25 µM) significantly reduced LPS-induced NF-κB activation, as determined by the quantification of p65 phosphorylation, and SWCNT-induced activation of NF-κB was also blocked by Pepinh-MYD, but not by Pepinh-Control (25 µM) (Fig.5b). Moreover, inhibition of MyD88 impeded chemokine production in cells exposed to SWCNTs. Thus, Pepinh-MYD (25 µM) significantly reduced LPS-induced CCL5 secretion, and SWCNT-triggered release of CCL5 was also reduced by Pepinh-MYD, but not by the control peptide (Fig.5c). These data thus provided evidence for TLR2/4-MyD88-NF-κB signaling in SWCNT-induced chemokine production in human macrophages. SWCNTs are internalized by HMDM at 24 h (Fig.S2) and we recently provided evidence that macrophage uptake of SWCNTs may occur already after a few hours24. To assess whether cellular uptake of SWCNTs is required for chemokine responses, we determined the secretion of CCL5 in HMDM exposed to SWCNTs (30 µg/ mL) following preincubation with or without cytochalasin D (10 µM), an inhibitor of actin polymerization that blocks endocytosis24. As shown in Fig.5d, cytochalasin D did not affect LPS-induced or SWCNT-induced production of CCL5, suggesting that this event is relayed via cell surface signaling. We also cultivated macrophages in medium with or without 10% FBS in order to test whether the effect of SWCNTs on chemokine secretion was influenced by the presence of serum proteins. As shown in Fig.S5b, SWCNT-triggered production of CCL5 was comparable in the presence or absence of serum. LPS was included as a positive control. To confirm the findings obtained in monocyte-derived macrophages, and in order to address the role, if any, of specific TLRs for the ‘sensing’ of SWCNTs, we used HEK293 cells stably transfected with human TLR2 or TLR4 and an NF-κB-inducible reporter gene25. Furthermore, in order to ascertain whether SWCNTs are capable of TLR activation per se, or whether the interaction is due to serum proteins adsorbed on the surface of the nanomaterials26, we performed the experiments in reporter cell lines cultured in medium supplemented or not with 10% fetal bovine serum (FBS). LPS (100 ng/mL) was used as a positive control. LPS triggered pronounced activation of TLR4 and a significant activation of TLR2 (Fig.6a). Interestingly, SWCNTs also activated TLR4 and, to a lesser degree, TLR2, and the level of activation in the presence and absence of FBS was indistinguishable. The latter finding suggested that SWCNTs are sensed directly by TLRs (Fig.6b). Functional role of chemokine secretion. Chemokines (Greek: kinos, movement) are named for their role in inducing directed migration or chemotaxis in neighboring responsive cells. We therefore addressed whether the conditioned medium of macrophages exposed to SWCNT versus GO would act as a chemoattractant for other immune cells. Considering that CCL3 and CCL5 are the most effective chemoattractants for immature DCs27, we studied cell migration using primary human monocyte-derived DCs versus non-differentiated primary human monocytes. Surface expression of the chemokine receptor, CCR5 was higher for DCs than monocytes (Fig.7a), Figure 4. SWCNT-triggered chemokine secretion is NF-κB-dependent. Pretreatment with the NF-κB inhibitor, Bay 11-7082 (10 µM) of HMDM exposed to 30 µg/mL SWCNTs or medium alone reduced the secretion of CCL3 (a) and completely blocked the secretion of CCL5 (b), by multi-plex assay. Data are mean values ± S.D. of three independent experiments using cells from different donors; p-values by Student’s t-test, * < 0.05; *** < 0.001. www.nature.com/scientificreports/ 7 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 in line with published results28. Furthermore, conditioned medium of SWCNT-exposed HMDM was a weak chemoattractant for monocytes, but promoted the migration of DCs (p < 0.05) (Fig.7b). Thus, the conditioned medium served as a chemoattractant for cells expressing CCR5 and did not remarkably affect the migration of cells that expressed low levels of CCR5. However, conditioned medium of GO-exposed HMDM did not promote cell migration (Fig.7b), in line with the observation that only SWCNTs triggered chemokine secretion. Molecular docking studies. Previous theoretical studies have suggested that C60 fullerenes and CNTs may block K+ channels29,30. In addition, recent computational studies suggested that the internal hydrophobic pockets of some TLRs might be capable of binding carbon-based nanostructures31. We performed molecular modelling of the TLR4:CNT complex to further elucidate how TLRs can interact with the CNTs; the studies were done in the absence of a protein corona. To better reproduce the experimental conditions, both pristine and carboxylated CNTs were modelled. The O:C atom ratio corresponding to the experimental zeta potential was set to 0.1532, which resulted in 84 carboxyl groups. Docking simulations of pristine CNTs gave rise to a unique cluster of very similar poses within the pre-defined energy range (10 kcal/mol). Conversely, carboxylated CNTs showed diverse binding modes, with interaction free energies differing only slightly (~1 kcal/mol). The best scoring binding mode, observed in both pristine and carboxylated CNTs, revealed two regions in TLR4, one interacting with the tip of the CNT and another in contact with its side-walls (Fig.8a,b). While the first region is localized in a highly hydrophobic area, which encompasses residues from Ile108 to Asn265, the second is found in the loops around Ile412 and Leu434 (Fig.9a,b). In oxidized CNTs, His159 and Arg264 are also within a distance to the carboxyl groups that is compatible with salt bridge formation. Alternatively, in the second region, Arg382, His431 and His458 are close enough to form ion-pair interactions with carboxyl groups on the CNT side-walls. Overall, the best binding mode is essentially guided by hydrophobic contacts between TLR4 and CNTs, but in the case of carboxylated CNTs the intermolecular interaction is strengthened by short-range electrostatics. The second and third top binding modes of carboxylated CNTs correspond to a completely different configuration, in which side-walls are in close contact with a large portion (from residue 87 to 289) of the TLR parallel beta-sheet pattern Figure 5. TLR2/4and MyD88-dependent secretion of chemokines. (a) Significant reduction of CCL5 secretion in HMDM after 12 h exposure to SWCNT (30 µg/mL) in the presence of the TLR2/4 inhibitor, oxPAPC (30 or 60 µg/mL). oxPAPC also blocked CCL5 secretion triggered by LPS (0.1 µg/mL) in a dosedependent manner. Furthermore, the MyD88 inhibitor, Pephinh-MYD (25 µM), but not Pepinh-Control (25 µM), reduced NF-kB p65 phosphorylation (b) and CCL5 expression (c) in cells exposed to SWCNT (30 µg/ mL) for 12 h. Pephinh-MYD (25 µM) also reduced NF-kB activation and CCL5 secretion by LPS (0.1 µg/mL). NF-kB p65 phosphorylation and CCL5 expression was determined by ELISA. (d) Cytochalasin D (10 μM), an inhibitor of actin polymerization, does not affect CCL5 secretion in HMDM exposed for 12 h to SWCNT (30 µg/ mL). LPS (0.1 µg/mL) was included as a control. CCL5 levels were determined by ELISA. Data shown in panels a to d are reported as mean values ± S.D. of at least three independent experiments using cells from different donors. p* < 0.05; ** < 0.01; *** < 0.001 (one-way ANOVA with post-hoc tukey’s test). www.nature.com/scientificreports/ 8 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 in the inner part of the protein (Fig.8c–f, Fig.9a,b). Interestingly, the area of interaction of these poses intersects one of the two regions already observed for the best pose. This finding is consistent with the negligible energy difference of these binding modes with respect to the best scoring mode. Nevertheless, as opposed to the latter, these two classes of poses are clearly dominated by electrostatic interactions, as shown by the distances between the carboxyl groups and some charged residues (His179, Arg257, Arg289, Arg355), which are compatible with the presence of stabilizing ion-pair interactions. Discussion The innate immune system does not respond to microbes in a nonspecific manner; in fact, pathogen recognition by the innate immune system is specific, relying on PRRs that have evolved to detect molecular signatures known as PAMPs21. Thus, a relatively small number of immune receptors are employed by macrophages and other immune cells to detect a vast array of microorganisms; it is intriguing to speculate that similar principles or mechanisms might be deployed for immune recognition of various classes of nanoparticles33. However, to date, there are few if any examples of specific immune sensing of engineered nanomaterials and the problem is confounded by a number of factors. First, many studies are performed using nanoparticles that are not well Figure 6. SWCNTs trigger TLR2 and TLR4 activation. (a) HEK 293 cells co-transfected with human TLR2 (HEK-Blue™ hTLR2) or TLR4 (HEK-Blue™ hTLR4 cells) and an NF-κB/AP-1-secreted embryonic alkaline phosphatase (SEAP) reporter gene were exposed to SWCNT (30 µg/mL) for 12 h in the presence or absence of 10% FBS. LPS (0.1 µg/mL) was included as a positive control. SWCNTs activated TLR2/4 independently of the presence of serum in the culture medium. Data are shown as mean values ± S.D. of three independent experiments. (b) Schematic diagram showing the ‘sensing’ of SWCNTs by HMDMs via TLR receptors resulting in MyD88-dependent activation of NF-kB leading to nuclear translocation of NF-kB with transcription and secretion of CCL5. The secreted chemokine(s) induce chemotaxis of immune cells bearing the corresponding receptor(s). Figure 7. Macrophage secreted factors promote migration of DCs. (a) Expression of the chemokine receptor CCR5 in primary human monocytes (Mo) and monocyte-derived dendritic cells (DCs) was determined by flow cytometry. The average expression of CCR5 in cells from three different donors is depicted. (b) Migration of monocytes (Mo) and DCs in response to conditioned medium (CM) of human monocyte-derived macrophages (HMDM) exposed to 100 µg/mL SWCNT (CM-SWCNT) or 100 µg/mL GO (CM-GO). Cell migration (3 h period) was determined by using transwell chemotaxis microchambers. Data are shown as mean values ± S.D. of three independent experiments; p* < 0.05, Student’s t-test. www.nature.com/scientificreports/ 9 SCIENTIFIC REPORTS | (2018) 8:1115 | DOI:10.1038/s41598-018-19521-9 characterized, or not uniform in appearance within the same sample, making it difficult to draw conclusions regarding specific properties of nanoparticles and their biological behavior34. In addition, nanoparticles are frequently contaminated with bacterial endotoxin, as may be the case for other biomaterials, leading potentially to erroneous results, especially when studying interactions with immune-competent cells17. Furthermore, nanoparticles are known to rapidly adsorb proteins and other biomolecules, and this is thought to endow the nanoparticles with a new, biological ‘identity’ such that these adsorbed biomolecules could dictate biological interactions: cells may not ‘see’ the pristine nanoparticle surfaces35. In addition to these considerations, it has been argued that there are no nano-specific (i.e., size-dependent) biological effects of nanoparticles, and therefore no novel effects are to be expected36. However, it is worth noting that many biological processes transpire at the nano-scale. Thus, it follows from this argument that nanoparticles, as a function of their small size, may interfere with biological processes in a manner not seen for larger particles37. Park et al.29 showed that purified SWCNTs blocked Figure 8. Molecular docking of CNTs and TLR4. Results of docking simulations of pristine and carboxylated CNTs and TLR4. (a) The best binding mode for pristine CNT. (b) best binding mode for carboxylated CNT. (c) lateral view of the second top binding pose for carboxylated CNT. (d) lateral view of the third top binding pose for the same CNT. (e) top view of the same configuration as in (c). (f) top view of the same configuration as in (d). 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Author Contributions S.P.M. and O.B. performed in vitro experiments and analyzed data; F.T.A. and T.B. contributed to in vitro experiments and data analysis; I.G. characterized the SWCNT and GO samples, supervised by S.M., E.K., M.B., and A.S. contributed to the material characterization of the SWCNT samples; P.C. and L.S. performed the molecular docking studies; H.S.M. and R.A. performed the microarray experiments, supervised by R.L., and P.K. conducted the downstream data analysis; B.F. conceived and coordinated the study, and wrote the paper with S.P.M. and O.B.; all authors approved the final version of the paper. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-19521-9. Competing Interests: The authors declare that they have no competing interests. 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