Carbon Nanomaterials Promote M1/M2 Macrophage Activation

www.small-journal.com 1907609 (1 of 12) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Full PaPer Carbon Nanomaterials Promote M1/M2 Macrophage Activation Pia Anneli Sofia Kinaret, Giovanni Scala, Antonio Federico, Jukka Sund, and Dario Greco* Dr. P. A. S. Kinaret, Prof. D. Greco Institute of Biotechnology Helsinki Institute of Life Science University of Helsinki Helsinki 00790, Finland E-mail: [email protected] Dr. G. Scala Faculty of Biological Sciences University of Naples Naples 80100, Italy Dr. A. Federico, Dr. J. Sund, Prof. D. Greco Faculty of Medicine and Health Technology Tampere University Tampere 33520, Finland The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smll.201907609. DOI: 10.1002/smll.201907609 effects are usually screened with relatively high doses, enabling evaluation of cell viability, apoptosis, ROS-production, and other biomarkers related to cellular stress.[4,5] Macrophages are among the first responders to foreign insults. They orchestrate immune responses by secreting inflammatory cytokines, which, in turn, help to recruit other immune cells. Moreover, they stimulate antigen-presenting cells (APC) to facilitate T-cell activation and adaptive immunity. Macrophages are extremely plastic cells, able to switch their phenotype after certain stimuli.[6,7] After encountering a foreign molecular pattern, macrophages produce a specific set of signals, thus determining the maturation and polarization status of the newly recruited immature cells such as monocytes. By modulating the expression of immune genes and secretion of cytokines, macrophages are able to adjust the microenvironment, boosting specific immune responses.[8] Even though the purpose of macrophages as central mediators of immune system is well recognized and has been extensively characterized, nanomaterial-mediated macrophage polarization and programming is still largely unknown.[9] CNM can induce inflammatory responses in vitro as well as in vivo.[10,11] Nevertheless, the dose and time-dependent immunomodulation of CNM is still largely missing. Instead of short, acute-phase toxicity studies, longer exposure time points need to be considered in order to understand changes in the cell phenotypes as well as the activation of adaptive immune response. This was demonstrated, for example, by Parise etal., who concluded that 48 h in vitro exposure is necessary to identify 22 sensitizers based on the altered gene expression levels in macrophages.[12] Possibly harmful nanomaterials with carefully adjusted, nontoxic doses can also exert immunomodulatory effects, by increasing or decreasing the activation of the immune system.[13] In year 2000, Mills etal. suggested a new classification of macrophages, including inflammation-promoting M1-type and healing M2-type macrophages.[14] Since then, based on activated biomarkers, several studies distinguishing different types of macrophage populations as well as suggesting new populations, such as M3-type and tumor-associated macrophages (TAM), have been published.[6] Widely accepted M1 markers include secretion and expression of TNF, IL-1β, Toxic effects of certain carbon nanomaterials (CNM) have been observed in several exposure scenarios both in vivo and in vitro. However, most of the data currently available has been generated in a high-dose/acute exposure setup, limiting the understanding of their immunomodulatory mechanisms. Here, macrophage-like THP-1 cells, exposed to ten different CNM for 48 h in low-cytotoxic concentration of 10 µgmL−1, are characterized by secretion of different cytokines and global transcriptional changes. Subsequently, the relationships between cytokine secretion and transcriptional patterns are modeled, highlighting specific pathways related to alternative macrophage activation. Finally, timeand dose-dependent activation of transcription and secretion of M1 marker genes IL-1β and tumor necrosis factor, and M2 marker genes IL-10 and CSF1 is confirmed among the three most responsive CNM, with concentrations of 5, 10, and 20 µgmL−1 at 24, 48, and 72 h of exposure. These results underline CNM effects on the formation of cell microenvironment and gene expression leading to specific patterns of macrophage polarization. Taken together, these findings imply that, instead of a high and toxic CNM dose, a sub-lethal dose in controlled exposure setup can be utilized to alter the cell microenvironment and program antigen presenting cells, with fascinating implications for novel therapeutic strategies. 1. Introduction Several types of carbon nanomaterials (CNM) are known to induce inflammatory responses in vitro and in vivo.[1–3] Toxic © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Small 2020, 16, 1907609 www.advancedsciencenews.com www.small-journal.com 1907609 (2 of 12) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim IL-6, IL-8, and IL-12.[8,15,16] M2 phenotype, on the other hand, is further divided into (at least) three subsets, M2a (Th2-type, killing), M2b (regulatory macrophages), and M2c (tissue remodeling, wound healing, matrix deposition). Expression or secretion of genes, such as TGF-β, CSF1, and DC-SIGN are considered M2-markers together with cytokines IL-10 and/or CCL18.[17,18] Here, we first exposed PMA-differentiated THP-1 macrophage-like cells to non-lethal dose of 10 µgmL−1 of ten CNM (Table1). After 48 h exposure, we examined the subsets of induced markers linked to classically activated M1and alternatively activated M2-type macrophages. We found distinct phenotypic markers to be regulated, suggesting that macrophages are able to modulate and program themselves depending on the CNM exposure, as well as to change their signaling cascades by time. To confirm a dose and time related polarization effect, we further validated applicable M1/M2 markers by RT-qPCR and U-Plex immunoassay with the three most provoking CNM: graphite nanofibers (GNF), rigid and long multiwalled carbon nanotubes (rCNT), and short multiwalled carbon nanotubes (Baytubes). Based on our previous results, we focused on three low-cytotoxic CNM concentrations (5, 10, and 20 µgmL−1), normalized across the different nanomaterials based on their phenotypic effects in vitro.[19] We focused on three long time points (24, 48, and 72 h), to ensure adequate presentation of all the tested nanoparticles with the cells as well as to optimize the observation of macrophage adaptation. 2. Results and Discussion 2.1. Macrophages Respond to CNM Exposure by Secreting Distinct Sets of Cytokines As previously reported, the concentration of 10 µgmL−1 used in this study is suitable to investigate fine immunomodulatory effects of CNM, for it does not significantly impact on the cell viability and metabolism (Figure S3 in Scala etal.[19]). Similar results on MWCNT-induced cytotoxicity have been shown also before, with concentrations ranging between 1 and 10 µgmL−1 on THP-1 and BEAS-2B cells.[20] We first focused on the possibility that CNM exposure would trigger patterns of cytokine secretion. For this, IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-17, IFNγ, and TNF were quantified in the cell culture supernatant after 48-h exposure. Seven out of ten nanomaterials in the panel did not induce any significant cytokine secretion above the detection limits of the assay. Thus, we further focused on graphite nanofibers (GNF), rigid and long multiwalled carbon nanotubes (rCNT), and short multiwalled carbon nanotubes (Baytubes), which exerted detectable increase in cytokine secretion (Figure1A). Based on these results, GNF was the only material in the panel inducing strong secretion of acute-phase pro-inflammatory tumor necrosis factor (TNF), a marker of classically activated macrophages (M1), usually induced by lipopolysaccharide (LPS) or pathogen stimuli. Also, the anti-inflammatory cytokine Interleukin 10 (IL-10) and the pro-inflammatory cytokine Interleukin 1β (IL-1β) was found to be significantly secreted after GNF exposure, suggesting acute phase response to GNF. rCNT exposure, on the other hand, did not trigger TNF secretion at 48 h exposure, but resulted in a sharp increase of pro-inflammatory cytokine IL-1β as well as anti-inflammatory cytokine IL-10. Absence of TNF secretion suggests, for example, inhibition through secretion of anti-inflammatory cytokine IL-10.[21,22] Baytubes instead, caused mild secretion of IL-10, but no significant secretion of the M1 cytokines IL-1β or TNF. All the remaining 7 CNM screened in this study did not exert any significant cytokine secretion. We could not identify an individual CNM intrinsic property that would straightforwardly explain cellular unresponsiveness, suggesting that several features, such as shape, aspect ratio, and surface area, affect, in combination, macrophage activation, as proposed also earlier.[23–25] 2.2. CNM Exposure Modulates Cytokine Transcription in Macrophages In order to test the hypothesis that the induced cytokine secretion would also correspond to induction of transcription, we further examined the cytokine changes at the transcriptional level (Figure 1B; Table S1, Supporting Information). We further focused on transcriptional responses caused by rCNT, Small 2020, 16, 1907609 Table 1. Carbon nanomaterials and their properties. Material name Producer Acronym Type Length [nm] Diameter [nm] Surface area [m2g−1] Aspect ratio Graphite nanofibers (Sigma) Sigma-Aldrich GNF Fiber 10000 140 32 71 Multiwalled carbon nanotube (Mitsui) Mitsui & Co. rCNT Tube 13000 50 22 260 Multiwalled carbon nanotube (Bayer) Bayer Material Science Baytubes Tube 1000 15 204 69 Carbon black (Evonik) Evonik Industries/Degussa CBL Particle 14 14 265 1 Fullerene C60 (MTR) MTR Ltd. FUL Sphere 100 100 20 1 Single-walled carbon nanotube (Sigma) Sigma-Aldrich SIG_SW Tube 50000 1 567 45450 Single-walled carbon nanotube (SES) SES research SES_SW Tube 1500 2 436 750 Multiwalled carbon nanotube (SES) SES research SES_MW Tube 1500 20 60 75 Multiwalled carbon nanotube (Cheaptubes) Cheaptubes Inc. CHT Tube 30000 12 180 2600 Multiwalled carbon nanotube (Sigma) Sigma-Aldrich SIG_MW Tube 100000 15 119 6660 www.advancedsciencenews.com www.small-journal.com 1907609 (3 of 12) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GNF, and Baytubes, since they elicited measurable, significant changes also in cytokine secretion. In the rCNT-exposed macrophages, both secreted cytokines IL-10 and IL-1β appeared to be also upregulated at the transcriptional level. TNF secretion was, in fact, strongly activated by GNF, but interestingly no transcriptional level activation was noted after 48 h exposure. As stated previously, the strong IL-10 secretion in case of GNF exposure might diminish the acute phase response by suppressing the production of TNF, triggering a negative feedback loop. Significant downregulation of TNF was, on the other hand, appreciated after exposure to Baytubes. Similarly, IL-10 was secreted in response to GNF exposure, but downregulation was detected after 48 h at the transcriptional level. Reduced expression suggests weakened inflammation, mediated by the action of anti-inflammatory cytokines such as IL-10, as suggested also in other studies.[7,26,27] On the other hand, we found IL-1β to be significantly secreted as well as transcribed after 48 h exposure to GNF. This is expected, for IL-1β is known to be regulated through an autocrine positive loop.[28,29] In our experimental model, the PMA-differentiation might be able to trigger IL-1β secretion, but does not seem to affect our observations at 48 h, since in Baytubes exposure, the IL-1β expression was found significantly downregulated, and no secretion was detected even though Baytube-exposed cells were treated similarly with PMA before the exposures. In addition, Baytubes caused only mild secretion of IL-10, but no transcriptional activation of the anti-inflammatory cytokine, suggesting mild, acute phase response with immunosuppressive effect at 48 h. In addition, we observed transcriptional induction of IL-6 by rCNT and GNF, but no detectable secretion (Figure1A,B). The differences between secretion and expression suggest that the changes in the microenvironment, probably rapidly established upon exposure as an acute response, sustain cell activation also after 48 h. In order to clarify the discrepancies in secretion and expression patterns, a detailed screening of time-related kinetic responses could be the focus of further studies. Discrepancies in secretion and expression patterns are also important to consider when investigating longer in vitro exposures. In some cases, also nanomaterial-related assay interference can result in inconsistent cytokine expression and secretion measurements. For example, cytokine adsorption to the CNM surface could be speculated as a reason for undetected IL-6, as suggested by Dilger etal.[30] However, as described in Section 2.5, we noted consistent dose-dependent secretion patterns of the cytokines of our interest, suggesting that this could be a marginal problem in our experimental setup. Alterations between cytokine secretion and gene expression in the same macrophage population after exposure advocates the possibility that by time macrophages adapt in a specific microenvironment by adjusting the polarization status toward M1 or M2 type. 2.3. Altered Pathways Reflect Macrophage Activation and Suggest Macrophage Polarization We next hypothesized that the establishment of different cytokine-driven microenvironments could further contribute to distinct expression patterns in macrophages, thus facilitating their polarization toward M1, M2, and mixed M1/M2 types. Thus, the differentially expressed genes from microarray analysis (p-value < 0.05, |logFC|>0.58) were characterized in search of over-represented biological functions (Figure2; Tables S1 and S2, Supporting Information). In order to recognize unique responses to distinct CNM, we compared the transcriptomic alterations caused by GNF, Baytubes, and rCNT against those produced by the other materials in our screening. We found 128, 74, and 215 differentially expressed unique genes due to GNF, Baytubes, and rCNT, respectively (“Exclusive genes” sheet in Table S2, Supporting Information). Based on the over-represented Gene Ontology terms, the exclusive gene sets were underlining M1/M2 status (Figure2). GNF activated several pro-inflammatory and IL-1βrelated pathways (Figure 2A; “GNF exclusive pathways” sheet in Table S2, Supporting Information), Baytubes elicited TGFβ2 production (Figure2B; “Baytubes exclusive pathways” sheet in Table S2, Supporting Information), whereas rCNT triggered chemotaxis and cytokine-mediated pathways (Figure2C; “rCNT exclusive pathways” sheet in Table S2, Supporting Information). When studied at the level of activated pathways and functional annotations, GNF, rCNT, and Baytubes showed interesting associations to macrophage polarization (Figure 2; Table S2, Supporting Information). For example, pathways Small 2020, 16, 1907609 Figure 1. Expression and secretion patterns of the ten CNM of 48-h exposure: A) significant (p-value 0.05) cytokine secretion denoted with red color; B) significant (p-value 0.05) expression denoted with red as upregulated, green as downregulated. Grey areas are indicating no significant changes in secretion or expression. www.advancedsciencenews.com www.small-journal.com 1907609 (4 of 12) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim related to glucocorticoid response were found to be associated only to rCNT exposure (Table S2, Supporting Information). Glucocorticoids are included in the M2-macrophage category, having anti-inflammatory and immunosuppressive effects.[31] GNF-enriched pathways contained numerous cholesterolrelated pathways (“GNF enrichments” sheet in Table S1, Supporting Information). Macrophages promote atherosclerosis and plaque formation by maintaining pro-inflammatory microenvironment.[17] Imbalance in cholesterol homeostasis increases the risk of atherosclerosis.[32] Interestingly, the cholesterol transporter genes ABCA1 and ABCG1 (Table2), both associated to atherosclerosis,[32] were found significantly upregulated after GNF exposure and are also found to be activated in ironrelated macrophage polarization, namely hemoglobin-associated macrophage (M(Hb)).[17,33–35] Furthermore, the expression of CCL5, secreted by M1-macrophages, was induced exclusively after GNF exposure (Table2). CCL5 is an expressed feature of inflammatory disorders such as atherosclerosis.[36,37] Unique, differentially expressed genes (DEGs) associated solely to GNF further supported the outcome, by activating pathways related Small 2020, 16, 1907609 Figure 2. Biological Process Gene Ontologies enriched by exclusive genes relative to a) GNF, b) Baytubes, and c) rCNT exposures. Bars are sorted by log of the p-value from the Fisher’s exact test multiplied by the z-score of the deviation from the expected rank. Statistics and lists of all exclusive genes and gene ontologies are reported in Table S2, Supporting Information. www.advancedsciencenews.com www.small-journal.com 1907609 (5 of 12) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim to IL-1β production and secretion (Figure 2; Table S2, Supporting Information). Baytubes-associated top-ranked, enriched pathways instead included immunity and virus-related pathways such as response and defense response to viruses, including influenza A, hepatitis C, herpes simplex infection, and measles, thus suggesting possible sensing mechanisms against viruses (“Baytubes enrichments”-sheet in Table S1, Supporting Information). It could be speculated that the size of Baytubes is reflecting certain size components of some viruses. This is also supported by the enriched oligoadenylate synthetase pathway. Oligoadenylate synthetases are antiviral enzymes that degrade viral and host RNA.[38] It has been shown that viruses can trigger M2-type activation and IL-10 expression, for example, in case of swine fever virus, hepatitis C, herpesviruses, and measles virus.[39–43] 2.4. Transcriptional Alterations Suggest Macrophage Polarization In order to understand the specific alterations in macrophage activation status, genes known to be associated to M1 and M2 type macrophages were more closely examined (Table2). Classically activated M1-type macrophages secrete a set of pro-inflammatory cytokines such as TNF, CXCL8, IL-1β, IL-6, IL-12, and IL-23.[8] Based on the transcriptional alterations, GNF induced pro-inflammatory, M1-type of macrophage polarization by inducing secretion of TNF and IL-1β as well as the expression of CXCL8, IL-6, and Il-1β. Furthermore, CCL5, secreted in high concentrations by M1 macrophages,[37] was differentially expressed only after GNF exposures. rCNT triggered altogether the strongest response by activating several inflammatory genes such as IL-1β, TRAF1, SOCS3, IL-24, and CXCL8 as well as stimulated the secretion of IL-1β and IL-10. Furthermore, the following M2-marker genes were found to be differentially expressed: IL-4I1, IL-10, CCL18, IL-1RN, CSF1 (M-CSF), ARG2, VEGFA, MMP9, CXCL2. The patterns of transcriptional regulation observed after rCNT exposure are compatible with both M1 and M2 types of activation, M2-type being more pronounced (Table2). rCNT induced the expression of cytokine IL-6, which is active in classically activated (M1) and alternatively activated (M2) macrophages.[17] Also, IL-10 and CCL18, known to be active in M2-macrophages, were overexpressed after rCNT exposure. Moreover, IL-4I1 gene, a novel regulator of M2 polarization, was found to be significantly induced by rCNT.[44] Il-1RN, an M2-cytokine that competes for the same receptors with IL-1β, was similarly highly expressed after rCNT exposure, suggesting yet another mechanism of inhibiting the pro-inflammatory responses. In addition, NF-κB inhibitor (NFKBIA), an inflammation regulator was significantly upregulated at 48 h rCNT exposure. NFKBIA activation suggests inhibition of transcription factor NF-κB, a key regulator in infection-related immune responses at the 48 h exposure. Instead, IFNγ-activated adhesion molecule (ICAM1), considered as M1-type marker,[31] was strongly induced by rCNT exposure. We have previously shown that rCNT activates Th2type of response in mouse lung by promoting secretion of Th2 cytokines IL-4, IL-5, and IL-13.[45,46] M2a-type macrophages stimulated by the aforementioned cytokines, are further able to express, for instance, IL-1RN (Anakinra), IL-10, TGF-β, and CSF1.[8] We found the same molecules induced after 48 h exposure to rCNT in vitro. The CSF1 gene, which leads to homeostatic or anti-inflammatory M2-like phenotype,[31,47] was highly expressed in rCNT-exposed macrophages. Regulatory (M2b) macrophages, have unbalanced levels of IL-10 and IL-12, downregulating IL-12 and producing IL-10 at the same time.[16,17] This was evident also in our experiments, as rCNT induced IL-10 but downregulated IL-12A. Regulatory M2-type macrophages are potent inhibitors of inflammation, even though they might retain the ability to produce also pro-inflammatory cytokines.[16] Along the same lines, also our results suggest that rCNT might cause mixed and unbalanced macrophage phenotypes triggering M2 activation as well as classically activated M1 macrophages. This was also concluded by Meng etal. with mouse macrophage cell line RAW264.7 exposed to MWCNT for 24 h, revealing M1/M2 mixed status.[48] By following our previous finding that rCNT can cause unconventional Th-2 type of allergic response in vivo,[46] it can be hypothesized that in complex tissues, with multiple cell types sharing the same microenvironment, a clearer M2-type response can be achieved, where Th2-type signaling molecules are more prominently induced. Exposure to Baytubes triggered secretion of IL-10 but resulted in significant downregulation of pro-inflammatory cytokines TNF and IL-1β. Instead, upregulation of M2-cytokines Small 2020, 16, 1907609 Table 2. Genes related to phenotypic markers of macrophages. Genes with Log2 fold change (red upregulated, green downregulated) and their corresponding p-values are presented. Genes logFCp.value logFCp.value logFCp.value ABCA10,009 <0,001 0,890 ABCG10,005 <0,001 0,005 ARG2 0,004 0,0510,011 CCL180,006 0,22 20 ,210 CCL5 0,262 0,00 50 ,001 CLEC12b 0,002 0,0080,192 CXCL20,054 0,31 40 ,658 CXCL3<0 ,001 0,16 70 ,462 CXCL8 (IL-8) 0,000 0,06 00 ,244 DC-SIGN0,976 0,48 00 ,208 ICAM1<0,001 0,04 00 ,032 IFNy 0,525 0,33 10 ,516 IL-10<0,001 0,0230,075 IL-12 (A)0,058 0,5680,019 IL-1a0,001 0,00 50 ,019 IL-1b<0,001 <0,001 <0,001 IL-1RN <0,001 0,17 70 ,061 IL-200,048 0,76 80 ,825 IL-230,173 0,55 30 ,457 IL-24<0,001 0,22 50 ,640 IL-4 0,469 0,48 90 ,952 IL-5 0,090 0,0380,712 IL-6 0,022 0,0270,181 IL4I1<0,001 0,2670,001 M-CSF0,001 0,0490,067 MMP9 0,001 0,05 00 ,702 NFKB10,002 0,29 50 ,116 NFKB20,008 0,1650,010 NFKBIA <0,001 0,1260,038 SOCS3<0,001 0,10 30 ,127 TGFB10,864 0,52 10 ,747 TGFB20,005 0,04 20 ,001 TLR8 0,044 0,0270,097 TNF0,047 0,151<0,001 TNFAIP3<0,001 <0,001 0,002 TNFAIP6<0,001 0,20 30 ,236 TNFAIP80,002 0,00 20 ,003 TRAF10,022 0,9280,014 VEGFA<0,001 0,0060,067 rCNT GN FB ay www.advancedsciencenews.com www.small-journal.com 1907609 (6 of 12) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim TGF-β2 and IL-4l1 was observed. TGF-β is known to activate M2a and M2c-types of macrophages,[17,49] whereas IL-4I1 is a novel regulator of M2 polarization.[44] Expression of IL-4l1 and strong downregulation of pro-inflammatory cytokines IL-1β and TNF suggest M2 polarization after Baytubes exposure. On the other hand, IL-10 expression was not detected despite its mild secretion was noted. Secretion of IL-10 suggests wound healing or regulatory M2 type of activation. According to Italiani and Boraschi, M2 macrophages in vitro can be characterized by high levels of IL-10 and TGF-β, and low levels of IL-12 and IL-23.[47] TGF-β is believed to play a role in alternative macrophage activation.[49] While we could observe IL-10 secretion, upregulation of TGF-β2 expression, and down regulation of IL-12A after Baytubes exposure, no significant regulation of IL-23 expression was observed. We compared our findings on the selected M1/M2 genes reported in Table2 with the results from previous studies carried out on murine and human primary monocyte derived macrophages, treated either with LPS (M1 activation) or with IL-4 (M2 activation).[50–53] The vast majority (31/39) of the genes reported in our panel (Table2) have a consistent expression pattern and are identified as M1 or M2 markers also in the complementing studies. For example, CCL5, reported to be strongly activated by LPS, indeed is activated by GNF but not the other CNMs in our screening. Likewise, IL-1b was induced by LPS treatment, similarly to the GNF and rCNT exposures. Overall, more commonalities in gene expression patterns were not surprisingly noted between LPS-treated human monocyte derived macrophages, GNFand rCNT-exposed macrophage-like THP-1 cells (Table S3 and Figures S1 and S2, Supporting Information). Taken together, our observations on the secretion patterns of cytokines and transcriptional alterations suggest that GNF might induce macrophage M1 type activation, Baytubes may promote M2 type of activation, while rCNT might exert a hybrid M1/M2 macrophage phenotype. 2.5. CNM Exert Macrophage Polarization in a Doseand Time-Dependent Manner Our observations with 48-h exposure suggest that macrophages stimulated with GNF, rCNT, or Baytubes develop distinct microenvironments, which are able to further induce macrophage polarization. To confirm timeand dose-related effect on the possible polarization, we chose IL-1β and TNF genes to illustrate M1-type activation and IL-10 and CSF1 to signify M2-type of macrophage polarization. We measured the changes in secretion and expression of the four marker genes with three different doses of 5, 10, and 20 µgmL−1 at three different exposure time points of 24, 48, and 72 h. GNF-exposed cells secreted and expressed IL-1β and TNF especially after 24and 72-h exposures, whereas M2 markers (IL-10 and CSF1) were diminished in a time-dependent manner (Figure3). Moreover, no IL-10 expression was observed at any of the measured time points, whereas secretion was progressively decreasing in time. This suggests that IL-10 might be an earlier, acute response before the first measured 24-h time point, and thus shows degradative pattern instead of active secretion. rCNT instead showed clear timeand partly dose-dependent accumulation of all four markers (Figure4). Interestingly, Baytubes activated expression and secretion of M2-related IL-10 after 72 h. CSF1 expression was also activated after 72 h with 10 and 20 µgmL−1, but no significant secretion was detected with any timepoint or concentration (Figure5). Secretion of IL-1β was observed at all concentrations and timepoints, but no expression was detected, suggesting again earlier, acute response diminishing the expression before the first measured time point of 24 h. Since in our experimental setup new monocytes are not introduced to the culture and the initial state is the same, these results suggest that macrophages are able to program and polarize themselves, depending on the exposure and the cytokines present in their local environment. This agrees with the study by Tarique et al. where the ability of human macrophages to reprogram or depolarize after alternative stimuli was tested.[18] Also, Lugo-Villarino et al. and Huang et al. suggested similar changes in macrophage polarization due to Mycobacterium tuberculosis or HIV infection.[54,55] These results suggest that, after nanoparticle contact, macrophages secrete certain sets of cytokines as an acute response which, together with signals coming from stressed or apoptotic cells as well as the nanoparticles themselves, trigger the macrophages to redesign themselves in time. Nonetheless, it should be considered that M1/M2 classification is oversimplified and signatures from different macrophage populations do not exclude each other, but often co-exist resulting in mixed phenotypes that further depend on the microenvironment.[31] 2.6. Discussion Here we report the ability of three different CNM to induce phenotypic changes in a cell culture model of human macrophages. Macrophage polarization is a dynamic process that remains incompletely understood.[56] Environmental factors are able to initiate phenotypic changes in macrophages. These include distinct signals from microbial products, damaged cells, glucocorticoid hormones, apoptotic bodies, and immune complexes. Additionally, polarization can be adjusted by local microenvironmental conditions. A number of receptor-directed signaling pathways involving the modulation of distinct transcriptional regulatory machineries are known to be involved in macrophage polarization.[56–58] Although our observations are not granular enough to resolve the whole chain of molecular alterations involved in CNM polarization, we do observe suggestive molecular alterations. In particular, the transcriptional regulatory mechanisms mediated by NF-κB, STAT3 and HIF1/2 seem to be altered in our exposure setups. For example, we observed significant SOCS3, HIF-2a, and IL-10 expression after 48 h rCNT exposure. Additionally, NF-κB activation can be detected from the 48 h exposures to rCNT and Baytubes. GNF activated TLR8, which is upstream to MyD88 and NF-κB activation.[59] However, the activation of different pathways should be studied through receptor activation after a relatively short period of time, which is out of the scope of the current study. Small 2020, 16, 1907609 www.advancedsciencenews.com www.small-journal.com 1907609 (7 of 12) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Dosimetric considerations are important when comparing the effects of ENM in vitro. We based the selection of the doses used in this study on their biological effects. We have previously characterized the cytotoxic potential of the same 10 CNM (Figure S3 in Scala et al.[19]) on THP-1 cells and concluded that a nominal dose of 10 µgmL−1 exerts no Small 2020, 16, 1907609 Figure 3. Secretion (pgmL−1) and expression (relative quantity, RQ) levels of IL-1β, TNF, Il-10, and CSF1 with GNF concentrations of 5, 10, and 20 µgmL−1 at 24, 48 and 72 h post-exposure compared to untreated control. www.advancedsciencenews.com www.small-journal.com 1907609 (8 of 12) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim significant cell death after 48 h exposure. For the subsequent experiments, we started from this observation and extended the range of the tested doses by halving and doubling the phenotypically characterized nominal dose of 10 µgmL−1. All the experiments reported in this study were conducted by plating the same number of cells (1000000 cells per well) Small 2020, 16, 1907609 Figure 4. Secretion (pgmL−1) and expression (relative quantity, RQ) levels of IL-1β, TNF, Il-10, and CSF1 with rCNT concentrations of 5, 10, and 20 µgmL−1 at 24, 48, and 72 h post-exposure compared to untreated control. www.advancedsciencenews.com www.small-journal.com 1907609 (9 of 12) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Small 2020, 16, 1907609 at ≈85% confluency. Based on the current literature about nanomaterial concentration selection, in vitro studies utilize higher concentrations typically ranging between 30 and 400 µg mL−1, usually for exposure times ranging between a few to 24 h maximum.[4,60,61] Our scope here was to test significantly lower doses at longer time points to induce Figure 5. Secretion (pg mL−1) and expression (relative quantity, RQ) levels of IL-1β, TNF, Il-10, and CSF1 with Baytube concentrations of 5, 10, and 20 µgmL−1 at 24, 48, and 72 h post-exposure compared to untreated control.