Role of hypoxia inducible factor 1α in cobalt nanoparticle induced cytotoxicity of human THP-1 macrophages

doi:10.12336/biomatertransl.2021.02.004

Biomaterials Translational ›› 2021, Vol. 2 ›› Issue (2): 143-150.doi: 10.12336/biomatertransl.2021.02.004

• RESEARCH ARTICLE • Previous Articles Next Articles

Role of hypoxia inducible factor 1α in cobalt nanoparticle induced cytotoxicity of human THP-1 macrophages

Wendy Rachel Francis^1,#, Zhao Liu¹^,^2,#, Sian E Owens¹, Xiao Wang¹^,³, Huaming Xue⁴, Alex Lord¹, Venkateswarlu Kanamarlapudi¹, Zhidao Xia¹^,^*()

1 Centre for Nanohealth, Swansea University Medical School, Swansea, UK
2 Department of Orthopaedic Surgery, Orthopaedic Institute, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China
3 Department of Orthopaedic Surgery, Johns Hopkins School of Medicine, Baltimore, MD, USA
4 Department of Orthopaedics, Yangpu Hospital affiliated to Tongji University School of Medicine, Shanghai, China.

Received:2021-01-26 Revised:2021-06-07 Accepted:2021-06-09 Online:2021-06-28 Published:2021-06-28
Contact: Zhidao Xia E-mail:z.xia@swansea.ac.uk
About author:#Author Equally.

Abstract

Abstract:

Cobalt is one of the main components of metal hip prostheses and cobalt nanoparticles (CoNPs) produced from wear cause inflammation, bone lyses and cytotoxicity at high concentrations. Cobalt ions mimic hypoxia in the presence of normal oxygen levels, and activate hypoxic signalling by stabilising hypoxia inducible transcription factor 1α (HIF1α). This study aimed to assess in vitro the functional role of HIF1α in CoNP induced cellular cytotoxicity. HIF1α, lysosomal pH, tumour necrosis factor α and interleukin 1β expression were analysed in THP-1 macrophages treated with CoNP (0, 10 and 100 μg/mL). HIF1α knock out assays were performed using small interfering RNA to assess the role of HIF1α in CoNP-induced cytotoxicity. Increasing CoNP concentration increased lysosomal activity and acidity in THP-1 macrophages. Higher doses of CoNP significantly reduced cell viability, stimulated caspase 3 activity and apoptosis. Reducing HIF1α activity increased the pro-inflammatory activity of tumour necrosis factor α and interleukin 1β, but had no significant impact on cellular cytotoxicity. This suggests that whilst CoNP promotes cytotoxicity and cellular inflammation, the apoptotic mechanism is not dependent on HIF1α.

Key words: cobalt nanoparticle, cytotoxicity, hypoxia inducible factor, macrophages, TNFα

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Figures/Tables 5

Figure 1. Micrographs of cryo-SEM illustrates THP-1 macrophages and their phagocytosis of CoNP. (A) Cell morphology of control THP-1 cells. (B) Cell morphology of THP-1 cells at 24 hours after CoNP (100 μg/mL). (C) High magnification of normal SEM showing a macrophage in contact with CoNPs. (D) Backscatter SEM of the same cells in C, revealing high electron density particles, in particular one cluster of CoNPs not showing in C. (E) Highlighted area demonstrating the cluster of CoNP not showing by normal SEM. (F) Highlighted area demonstrating phagocytosis of the cluster of CoNPs that were engulfed and submerged within the macrophage cell membrane. Scale bars: 50 μm in A, B; 5 μm in C, D; 1 μm in E, F. CoNP: cobalt nanoparticle; SEM: scanning electron microscope.

Figure 2. The pH within THP-1 macrophages at 24 hours after CoNP incubation was measured using LysoSensor Yellow/Blue DND 160 probes. (A-H) The blue fluorescence indicates neutral pH within the cell (A, C, E, G), whilst the yellow fluorescence shows the acidity of the lysosomes following the engulfment of CoNP (B, D, F, H). With increasing CoNP dose, the numbers and acidity of lysosomes within the cell increased whilst the pH within the cell remained neutral. CoNP: cobalt nanoparticle; Ly: lysosomes.

Figure 3. Analysis of HIF1α expression and the effect of HIF1α knockout on cytotoxicity and inflammatory factors (TNFα and IL-1β). (A) THP-1 cells were lysed for protein extraction 24 hours following treatment with CoNP (10 and 100 μg/mL). HIF1α concentration was measured by ELISA Duoset?. (B) Western blot of HIF1α expression. (B1) HIF1α concentration was increased with CoNP dose. (B2) Knockdown of HIF1α using siRNA showed a significantly reduced production of HIF1α protein in response to CoNP. (C, D) TNFα (C) and IL-1β (D) concentration in THP-1 cells with CoNP treatment were measured in the culture supernatants using enzyme-linked immunosorbent assay Duoset? 24 hours following CoNP treatment (0, 10, 100 μg/mL). Both TNFα (C) and IL-1β (D) secretion increased with CoNP dose in the healthy and transfected macrophages. Both TNFα and IL 1β increased significantly following transfection in comparison to the control. Cultures transfected with HIF1α siRNA showed significant up-regulation of TNFα and IL-1β in comparison to the mismatched control. (E) Percentage viability of healthy and transfected THP-1 cultures treated with CoNP (0, 10, 100 μg/mL) as measured by neutral red staining. Viability significantly reduced with increasing CoNP dose within the control, mismatched control and HIF1α knock out. There were no significant differences between the mismatched control and cultures transfected with HIF1α small interfering RNA. Data are expressed as mean ± SE. All experiments were repeated twice or three times with consistent results. *P < 0.05, *P < 0.01 (Wilcoxon matched pairs test). CoNP: cobalt nanoparticle; ELISA: enzyme-linked immunosorbent assay; HIF1α: hypoxia inducible transcription factor 1α; IL-1β: interleukin 1β; TNFα: tumour necrosis factor α.

Figure 4. Schematic diagram. CoNP: cobalt nanoparticle; HIF1α: hypoxia inducible transcription factor 1α; ROS: reactive oxygen species; siRNA: small interfering RNA.

Additional Figure 1. Bright field and confocal images illustrate the phagocytosis and the immunocytochemical staining of hypoxia inducible transcription factor 1α (HIF1α) and caspase 3 (Casp3) of THP-1 macrophages in response to different dose of cobalt nanoparticles (CoNPs). It shows a dose dependent increases of HIF1α staining (arrows to the red coloured staining) with the increases of CoNP doses, and Casp3 staining (arrows to the green coloured staining) is in correlation with HIF1α. However, Casp3 is less pronanced when the doses of CoNPs are lower than 1 μg/mL. Scale bars: 20 μm.

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Role of hypoxia inducible factor 1α in cobalt nanoparticle induced cytotoxicity of human THP-1 macrophages

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