Influence of metallic particles and TNF on the transcriptional regulation of NLRP3 inflammasome-associated genes in human osteoblasts

Abstract

Introduction: The release of mature interleukin (IL-) 1β from osteoblasts in response to danger signals is tightly regulated by the nucleotide-binding oligomerization domain leucine-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome. These danger signals include wear products resulting from aseptic loosening of joint arthroplasty. However, inflammasome activation requires two different signals: a nuclear factor-kappa B (NF-κB)-activating priming signal and an actual inflammasome-activating signal. Since human osteoblasts react to wear particles via Toll-like receptors (TLR), particles may represent an inflammasome activator that can induce both signals.

Methods: Temporal gene expression profiles of TLRs and associated intracellular signaling pathways were determined to investigate the period when human osteoblasts take up metallic wear particles after initial contact and initiate a molecular response. For this purpose, human osteoblasts were treated with metallic particles derived from cobalt-chromium alloy (CoCr), lipopolysaccharides (LPS), and tumor necrosis factor-alpha (TNF) alone or in combination for incubation times ranging from one hour to three days. Shortly after adding the particles, their uptake was observed by the change in cell morphology and spectral data.

Results: Exposure of osteoblasts to particles alone increased NLRP3 inflammasome-associated genes. The response was not significantly enhanced when cells were treated with CoCr + LPS or CoCr + TNF, whereas inflammation markers were induced. Despite an increase in genes related to the NLRP3 inflammasome, the release of IL-1β was unaffected after contact with CoCr particles.

Discussion: Although CoCr particles affect the expression of NLRP3 inflammasome-associated genes, a single stimulus was not sufficient to prime and activate the inflammasome. TNF was able to prime the NLRP3 inflammasome of human osteoblasts.

Keywords: NLRP3 inflammasome; human osteoblasts; joint replacement; metallic particles; osteolysis.

Figure 1

Schematic overview of the experimental setup and the methods used. The figure was created on 10/18/2023 at 10:55 AM with Biorender (Agreement number: YG26QJZVPQ) (https://biorender.com).

Figure 2

Influence of short-term particle exposure on cell morphology of human osteoblasts and influence of particle incorporation on hyperspectral data. (Left) Combined fluorescence darkfield images of osteoblasts exposed to CoCr particles at different time points were taken with the CytoViva^® microscope system. The actin cytoskeleton of cells was stained with phalloidin (green fluorescence), and the nuclei were counterstained with DAPI (blue fluorescence). (Top right) To determine particle uptake, hyperspectral images (HSI) of CoCr particles in cell culture medium and of particles after incubation with cells were taken with the CytoViva^® microscope system, and the mean spectral profiles are shown in a spectral library plot. Bar: 50 µm; 100 µm (CoCr particles).

Figure 3

Gene expression analysis of markers of TLR signaling (A-D) and NLRP3 inflammasome (F-J) in human osteoblasts treated with CoCr (blue dots), TNF (light turquoise dots), or CoCr + TNF (dark blue dots) compared to the untreated control (J: orange triangle) and evaluation of translocation of NF-κB to the nucleus via NF-κB staining (E). (A-D, F-I) The total RNA of osteoblasts was isolated, and relevant genes were examined via qPCR. The results were calculated by the 2^-ΔΔCt method and normalized to the unstimulated control (dotted line; in heatmaps: 1). (J) Intracellular IL-1β protein of cell lysates lysates after 8 h, 24 h, and 72 h of stimulation was examined by ELISA and related to the total protein amount. The results are shown as individual values (one data point per donor) with median and interquartile ranges (A-D, F, G, J) or as median within the heatmaps (H, I,; n=4). Statistical significance was determined using the 2-way ANOVA and Bonferroni multiple comparison post hoc test (A-I) and Kruskal-Wallis test with Dunn’s multiple comparisons test (J): *p<0.05; **p<0.01; ****p<0.0001 (significance between single stimulations); ⁺p<0.05; ⁺⁺p<0.01; ⁺⁺⁺p<0.001 (significance between TNF and CoCr + TNF); ^ap<0.05; ^bp<0.01; ^cp<0.001; ^dp<0.0001 (significance to unstimulated control). Significant differences within a treatment between time points are not shown in the graphs. n.d., not detectable.

Figure 4

Total free radical concentration from the supernatant and lysed cells after 8 h and 24 h treatment with CoCr particles (blue dots), TNF (turquoise dots), or CoCr + TNF (dark blue dots) of cells relative to untreated control. Statistical significance was determined using the 2-way ANOVA and Bonferroni multiple comparison post hoc test (A): ****p<0.0001 (significance between single stimulations); ⁺⁺⁺p<0.001 (significance between LPS and CoCr + LPS); ^cp<0.001, ^dp<0.0001 (significance to unstimulated control). Significant differences within a treatment between time points are listed in the text and not shown in the graphs. (B) Free radical concentration in the supernatant and from lysed cells after 8 h and 24 h. Data in the table are presented as mean values ± SEM (n=4, one data point per donor).

Figure 5

Effects on lactate dehydrogenase (LDH) activity (A) and staining of ASC-speck formation (B). LDH activity was measured in supernatants of osteoblasts after exposure with CoCr (blue dots), TNF (light turquoise dots), CoCr+TNF (dark blue dots), CoCr+ATP (dark blue dots), and TNF+ATP (green dots). The results are shown as individual values (one data point per donor) with median and interquartile ranges (n=3). Statistical significance was determined using the 2-way ANOVA and Bonferroni multiple comparison post hoc test: ⁺p<0.05 (significance between TNF and CoCr + TNF); ^bp<0.01; ^dp<0.0001 (significance to unstimulated control); ^§§§§p<0.0001 (significance between CoCr and CoCr + ATP); ^XXXXp<0.0001 (significance between TNF and TNF + ATP); ^Op<0.05 (significance between CoCr + ATP and TNF + ATP). An ASC (green) staining was used to investigate the formation of NLRP3 inflammasome after stimulating osteoblasts with CoCr particles, TNF, and CoCr + TNF for 8 h. Unstimulated cells were included as negative controls. Osteoblasts treated with TNF and ATP served as positive control. The nuclei were stained with DAPI (blue). Bars: 50 µm; 100 µm.

Figure 6

Markers of inflammation. Gene expression and secretion of IL-6 and IL-8 in human osteoblasts treated with CoCr (blue dots), TNF (light turquoise dots), or CoCr + TNF (dark blue dots) compared to the untreated control (A, B). The total RNA of osteoblasts was isolated, and relevant genes were examined via qPCR. The results were calculated by the 2^-ΔΔCt method and normalized to the unstimulated control. The release of IL-6 (C) and IL-8 (D) was examined in the supernatants of exposed osteoblasts by ELISA and related to the total protein amount. The results are shown as individual values (one data point per donor) with median and interquartile ranges (n=4). Statistical significance was determined using the 2-way ANOVA and Bonferroni multiple comparison post hoc test (A, B) and Kruskal-Wallis test with Dunn’s multiple comparisons test (C, D): *p<0.05; ****p<0.0001 (significance between single stimulations); °°p<0.01; °°°°p<0.0001 (significance between CoCr and co-stimulation); ^ap<0.05; ^bp<0.01; ^cp<0.001; ^dp<0.0001 (significance to unstimulated control). Significant differences within a treatment between time points are listed in the text and not shown in the graphs.