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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 Ti/TiO2 nanotubular vs flat surface on IKKβ and IkB-α phosphorylation.Notes: (A) RAW 264.7 cells were allowed to adhere on the substrates for 24 hours prior to stimulation with 1 µg·mL−1 LPS for specified times. The concentration of phospho-IKKβ in cell lysates was analyzed by the ELISA technique. The data are expressed as mean ± SD. **P<0.01; ***P<0.001. (B) Fluorescent immunodetection of p-IkB-α in untreated and LPS (1 µg·mL−1, for 10 minutes) stimulated macrophages using specific anti-p-IkB-α antibody (red). Nuclei were stained with DAPI (blue). Scale bar represents 20 µm.Abbreviations: cpTi, commercial pure titanium; DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; OD, optical density; SD, standard deviation; vs, versus.
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f2-ijn-10-6455: Effects of Ti/TiO2 nanotubular vs flat surface on IKKβ and IkB-α phosphorylation.Notes: (A) RAW 264.7 cells were allowed to adhere on the substrates for 24 hours prior to stimulation with 1 µg·mL−1 LPS for specified times. The concentration of phospho-IKKβ in cell lysates was analyzed by the ELISA technique. The data are expressed as mean ± SD. **P<0.01; ***P<0.001. (B) Fluorescent immunodetection of p-IkB-α in untreated and LPS (1 µg·mL−1, for 10 minutes) stimulated macrophages using specific anti-p-IkB-α antibody (red). Nuclei were stained with DAPI (blue). Scale bar represents 20 µm.Abbreviations: cpTi, commercial pure titanium; DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; OD, optical density; SD, standard deviation; vs, versus.

Mentions: Following, we investigated the inhibitory effect of the nanotubular surface on NF-κB activation by assessing the phosphorylation level of IKKβ protein and the expression level of phospho-IkB-α (p-IkB-α), by ELISA studies and immunofluorescence analysis, respectively. As depicted in Figure 2A, the stimulation of cells with LPS resulted in a rapid phosphorylation of IKKβ on Ser 177/181, reaching a maximum intensity level after 10 minutes and decreasing almost to the basal level at the 30 minutes time point. Comparing with the reference material (cpTi), it is evident that the contact between RAW 264.7 cells and Ti/TiO2 surface leads to a significant inhibition of the LPS-induced phosphorylation of IKKβ at both tested poststimulation time points, that is, 10 and 30 minutes (P<0.001), as well as in the absence of the pro-inflammatory stimulus (untreated macrophages, P<0.01). Further, IkB-α phosphorylation was studied by immunofluorescence staining to investigate its involvement in the inhibitory effect of TiO2 nanotubes on pro-inflammatory activation of macrophages. The cytosolic expression of p-IkB-α was observed even at 5-minute poststimulation, and was visible up to 30 minutes (data not shown). Within 10 minutes of LPS stimulation, a high level of phosphorylated IkB-α was remarked onto the surface of cpTi, while the nanotubular surface maintained a low expression level of p-IkB-α in the cytoplasm but with a slight increase as compared with untreated cultures (Figure 2B). The suppression of IKKβ and IkB-α phosphorylation by surface nanotopography is an essential event that is associated with the subsequent inhibition of NF-κB translocation into the nucleus.


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 Ti/TiO2 nanotubular vs flat surface on IKKβ and IkB-α phosphorylation.Notes: (A) RAW 264.7 cells were allowed to adhere on the substrates for 24 hours prior to stimulation with 1 µg·mL−1 LPS for specified times. The concentration of phospho-IKKβ in cell lysates was analyzed by the ELISA technique. The data are expressed as mean ± SD. **P<0.01; ***P<0.001. (B) Fluorescent immunodetection of p-IkB-α in untreated and LPS (1 µg·mL−1, for 10 minutes) stimulated macrophages using specific anti-p-IkB-α antibody (red). Nuclei were stained with DAPI (blue). Scale bar represents 20 µm.Abbreviations: cpTi, commercial pure titanium; DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; OD, optical density; SD, standard deviation; vs, versus.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608594&req=5

f2-ijn-10-6455: Effects of Ti/TiO2 nanotubular vs flat surface on IKKβ and IkB-α phosphorylation.Notes: (A) RAW 264.7 cells were allowed to adhere on the substrates for 24 hours prior to stimulation with 1 µg·mL−1 LPS for specified times. The concentration of phospho-IKKβ in cell lysates was analyzed by the ELISA technique. The data are expressed as mean ± SD. **P<0.01; ***P<0.001. (B) Fluorescent immunodetection of p-IkB-α in untreated and LPS (1 µg·mL−1, for 10 minutes) stimulated macrophages using specific anti-p-IkB-α antibody (red). Nuclei were stained with DAPI (blue). Scale bar represents 20 µm.Abbreviations: cpTi, commercial pure titanium; DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; OD, optical density; SD, standard deviation; vs, versus.
Mentions: Following, we investigated the inhibitory effect of the nanotubular surface on NF-κB activation by assessing the phosphorylation level of IKKβ protein and the expression level of phospho-IkB-α (p-IkB-α), by ELISA studies and immunofluorescence analysis, respectively. As depicted in Figure 2A, the stimulation of cells with LPS resulted in a rapid phosphorylation of IKKβ on Ser 177/181, reaching a maximum intensity level after 10 minutes and decreasing almost to the basal level at the 30 minutes time point. Comparing with the reference material (cpTi), it is evident that the contact between RAW 264.7 cells and Ti/TiO2 surface leads to a significant inhibition of the LPS-induced phosphorylation of IKKβ at both tested poststimulation time points, that is, 10 and 30 minutes (P<0.001), as well as in the absence of the pro-inflammatory stimulus (untreated macrophages, P<0.01). Further, IkB-α phosphorylation was studied by immunofluorescence staining to investigate its involvement in the inhibitory effect of TiO2 nanotubes on pro-inflammatory activation of macrophages. The cytosolic expression of p-IkB-α was observed even at 5-minute poststimulation, and was visible up to 30 minutes (data not shown). Within 10 minutes of LPS stimulation, a high level of phosphorylated IkB-α was remarked onto the surface of cpTi, while the nanotubular surface maintained a low expression level of p-IkB-α in the cytoplasm but with a slight increase as compared with untreated cultures (Figure 2B). The suppression of IKKβ and IkB-α phosphorylation by surface nanotopography is an essential event that is associated with the subsequent inhibition of NF-κB translocation into the nucleus.

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