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Attenuation of the macrophage inflammatory activity by TiO₂ nanotubes via inhibition of MAPK and NF-κB pathways.

Neacsu P, Mazare A, Schmuki P, Cimpean A - Int J Nanomedicine (2015)

Bottom Line: Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α.Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked.However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Bucharest, Bucharest, Romania.

ABSTRACT
Biomaterial implantation in a living tissue triggers the activation of macrophages in inflammatory events, promoting the transcription of pro-inflammatory mediator genes. The initiation of macrophage inflammatory processes is mainly regulated by signaling proteins of mitogen-activated protein kinase (MAPK) and by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. We have previously shown that titania nanotubes modified Ti surfaces (Ti/TiO2) mitigate the immune response, compared with flat Ti surfaces; however, little is known regarding the underlying mechanism. Therefore, the aim of this study is to investigate the mechanism(s) by which this nanotopography attenuates the inflammatory activity of macrophages. Thus, we analyzed the effects of TiO2 nanotubes on the activation of MAPK and NF-κB signaling pathways in standard and lipopolysaccharide-evoked conditions. Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α. Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked. Following, by using specific MAPK inhibitors, we observed that lipopolysaccharide-induced production of monocyte chemotactic protein-1 and nitric oxide was significantly inhibited on the Ti/TiO2 surface via p38 and ERK1/2, but not via JNK. However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production. Altogether, these data suggest that titania nanotubes can attenuate the macrophage inflammatory response via suppression of MAPK and NF-κB pathways providing a potential mechanism for their anti-inflammatory activity.

No MeSH data available.


Related in: MedlinePlus

Effects of titania nanotubes vs cpTi on the nuclear translocation of NF-κB-p65.Notes: (A) Fluorescent imaging of p65 in untreated and LPS-stimulated RAW 264.7 macrophages. Scale bar represents 10 µm. (B) Percentage of cells displaying nuclear accumulation of p65. Comparison was made between Ti/TiO2 and cpTi at 10 and 30 minutes poststimulation on six representative microscopic fields. The data are expressed as mean ± SD. ***P<0.001.Abbreviations: cpTi, commercial pure titanium; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; SD, standard deviation; vs, versus.
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f3-ijn-10-6455: Effects of titania nanotubes vs cpTi on the nuclear translocation of NF-κB-p65.Notes: (A) Fluorescent imaging of p65 in untreated and LPS-stimulated RAW 264.7 macrophages. Scale bar represents 10 µm. (B) Percentage of cells displaying nuclear accumulation of p65. Comparison was made between Ti/TiO2 and cpTi at 10 and 30 minutes poststimulation on six representative microscopic fields. The data are expressed as mean ± SD. ***P<0.001.Abbreviations: cpTi, commercial pure titanium; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; SD, standard deviation; vs, versus.

Mentions: The activation of IKK phosphorylates the inhibitory IkB-α protein, leading to its degradation and the subsequent release of the NF-κB complex, which then translocates into the nucleus as to activate the transcription factors of the pro-inflammatory genes. To examine the NF-κB nuclear translocation, we performed immunocytochemistry studies on the p65 subunit of NF-κB. The fluorescent images show that for untreated macrophages, p65 was sequestered in the cytoplasm, whereas for LPS-stimulated macrophages p65 appeared in both the cytosol and the nuclear zone (Figure 3A). Furthermore, as shown in Figure 3B, LPS-induced a rapid nuclear accumulation of p65, peaking at 10 minutes and slowly decreasing in time. The percentage of cells displaying a NF-κB shift to the nucleus was significantly higher (P<0.001) on the flat Ti surface as compared with Ti/TiO2, for non-stimulated and LPS-stimulated (10 minutes and 30 minutes) macrophages. Moreover, a slight increase in the NF-κB nuclear accumulation upon LPS-stimulation was remarked on the nanotubular surface, while the cpTi surface elicited a marked enhancement of NF-κB nuclear translocation. Consequently, Ti/TiO2 nanotubes inhibited the nuclear translocation of the NF-κB p65 subunit, after the subunits were dissociated from IkB-α.


Attenuation of the macrophage inflammatory activity by TiO₂ nanotubes via inhibition of MAPK and NF-κB pathways.

Neacsu P, Mazare A, Schmuki P, Cimpean A - Int J Nanomedicine (2015)

Effects of titania nanotubes vs cpTi on the nuclear translocation of NF-κB-p65.Notes: (A) Fluorescent imaging of p65 in untreated and LPS-stimulated RAW 264.7 macrophages. Scale bar represents 10 µm. (B) Percentage of cells displaying nuclear accumulation of p65. Comparison was made between Ti/TiO2 and cpTi at 10 and 30 minutes poststimulation on six representative microscopic fields. The data are expressed as mean ± SD. ***P<0.001.Abbreviations: cpTi, commercial pure titanium; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; SD, standard deviation; vs, versus.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608594&req=5

f3-ijn-10-6455: Effects of titania nanotubes vs cpTi on the nuclear translocation of NF-κB-p65.Notes: (A) Fluorescent imaging of p65 in untreated and LPS-stimulated RAW 264.7 macrophages. Scale bar represents 10 µm. (B) Percentage of cells displaying nuclear accumulation of p65. Comparison was made between Ti/TiO2 and cpTi at 10 and 30 minutes poststimulation on six representative microscopic fields. The data are expressed as mean ± SD. ***P<0.001.Abbreviations: cpTi, commercial pure titanium; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; SD, standard deviation; vs, versus.
Mentions: The activation of IKK phosphorylates the inhibitory IkB-α protein, leading to its degradation and the subsequent release of the NF-κB complex, which then translocates into the nucleus as to activate the transcription factors of the pro-inflammatory genes. To examine the NF-κB nuclear translocation, we performed immunocytochemistry studies on the p65 subunit of NF-κB. The fluorescent images show that for untreated macrophages, p65 was sequestered in the cytoplasm, whereas for LPS-stimulated macrophages p65 appeared in both the cytosol and the nuclear zone (Figure 3A). Furthermore, as shown in Figure 3B, LPS-induced a rapid nuclear accumulation of p65, peaking at 10 minutes and slowly decreasing in time. The percentage of cells displaying a NF-κB shift to the nucleus was significantly higher (P<0.001) on the flat Ti surface as compared with Ti/TiO2, for non-stimulated and LPS-stimulated (10 minutes and 30 minutes) macrophages. Moreover, a slight increase in the NF-κB nuclear accumulation upon LPS-stimulation was remarked on the nanotubular surface, while the cpTi surface elicited a marked enhancement of NF-κB nuclear translocation. Consequently, Ti/TiO2 nanotubes inhibited the nuclear translocation of the NF-κB p65 subunit, after the subunits were dissociated from IkB-α.

Bottom Line: Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α.Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked.However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Bucharest, Bucharest, Romania.

ABSTRACT
Biomaterial implantation in a living tissue triggers the activation of macrophages in inflammatory events, promoting the transcription of pro-inflammatory mediator genes. The initiation of macrophage inflammatory processes is mainly regulated by signaling proteins of mitogen-activated protein kinase (MAPK) and by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. We have previously shown that titania nanotubes modified Ti surfaces (Ti/TiO2) mitigate the immune response, compared with flat Ti surfaces; however, little is known regarding the underlying mechanism. Therefore, the aim of this study is to investigate the mechanism(s) by which this nanotopography attenuates the inflammatory activity of macrophages. Thus, we analyzed the effects of TiO2 nanotubes on the activation of MAPK and NF-κB signaling pathways in standard and lipopolysaccharide-evoked conditions. Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α. Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked. Following, by using specific MAPK inhibitors, we observed that lipopolysaccharide-induced production of monocyte chemotactic protein-1 and nitric oxide was significantly inhibited on the Ti/TiO2 surface via p38 and ERK1/2, but not via JNK. However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production. Altogether, these data suggest that titania nanotubes can attenuate the macrophage inflammatory response via suppression of MAPK and NF-κB pathways providing a potential mechanism for their anti-inflammatory activity.

No MeSH data available.


Related in: MedlinePlus