Single-cell assessment of the modulation of macrophage activation by ex vivo intervertebral discs using impedance cytometry

Abstract

Measurement of macrophage activation and its modulation for immune regulation is of great interest to arrest inflammatory responses associated with degeneration of intervertebral discs that cause chronic back pain, and with transplants that face immune rejection. Due to the phenotypic plasticity of macrophages that serve multiple immune functions, the net disease outcome is determined by a balance of subpopulations with competing functions, highlighting the need for single-cell methods to quantify heterogeneity in their activation phenotypes. However, since macrophage activation can follow several signaling pathways, cytometry after fluorescent staining of markers with antibodies does not often provide dose-dependent information on activation dynamics. We present high throughput single-cell impedance cytometry for multiparametric measurement of biophysical changes to individual macrophages for quantifying activation in a dose and duration dependent manner, without relying on a particular signaling pathway. Impedance phase metrics measured at two frequencies and the electrical diameter from impedance magnitude at lower frequencies are used in tandem to benchmark macrophage activation by degenerated discs against that from lipopolysaccharide stimulation at varying dose and duration levels, so that reversal of the activation state by curcumin can be ascertained. This label-free single-cell measurement method can form the basis for platforms to screen therapies for inflammation, thereby addressing the chronic problem of back pain.

Keywords: Cytometry; Impedance; Inflammation; Intervertebral disc; Macrophages; Microfluidics.

Fig. 1.

A. Biological sample preparation, including: (i) ex vivo dissection of mouse lumbar intervertebral discs, (ii) stimulation with inflammatory cytokine IL-1β, and (iii) co-culture with macrophages in a 24-well plate (iv). B. Impedance cytometry of activated macrophages.

Fig. 2.

Heterogeneity in macrophage (Mac) phenotypes due to intercellular interactions as a function of cell seeding density. Scatter plot of impedance phase at 0.5 MHz (φZ_{0.5 MHz}) versus electrical diameter $\left(\sqrt[3]{{\left|Z\right|}_{0.5\text{MHz}}}\right)$ of macrophages is shown at different cell seeding densities: A. High (5 × 10⁵ cells/mL), B. Medium (2 × 10⁵ cells/mL), and C. Low (1 × 10⁵ cells/mL), with 0.5 mL per well. D. Histogram of φZ_{0.5 MHz} for control (untreated) macrophages at different cell seeding densities is compared to heat treated samples, to show stress rather than viability loss.

Fig. 3.

LPS dose and duration-dependent macrophage activation based on comparing control (untreated) macrophages to those treated with LPS doses of 10 ng/mL and 100 ng/mL for 6 h, and 100 ng/mL for 24 h. A. Scatter plot (and histograms) of φZ_{2 MHz} vs. φZ_{18 MHz} shows downshifting with activation. B. Histograms of electrical diameter; C. Mean electrical diameter shows significant differences between samples (***p < 0.001); D. Mean φZ_{2 MHz} shows significant differences between samples (**p < 0.01 level; ***p < 0.001); E. Mean φZ_{18 MHz} shows significant differences between samples (*p < 0.05 level; **p < 0.01 level; ***p < 0.001); F. Cross-validation based on secreted nitrite measured in culture media using the Griess assay. Polarized morphology of macrophages after 6 h LPS treatment is in SI: Fig. S9B.

Fig. 4.

Degenerated discs stimulate macrophages (Mac) towards a pro-inflammatory phenotype based on impedance metrics, NO secretion, and inflammatory gene expression; Mac with control discs, and Mac with degenerated disc (IL-1β 100 ng/mL, 2 days). A. Scatter plot (and histograms) of φZ_{2 MHz} vs. φZ_{18 MHz} shows downshifting with degenerated discs to indicate activation. B. Histograms of electrical diameter; C. Mean electrical diameter shows significant differences between samples (***p < 0.001); D. Mean φZ_{2 MHz} shows significant differences between samples (*p < 0.05 level; ***p < 0.001); E. Mean φZ_{18 MHz} shows significant differences between samples (*p < 0.05 level,***p < 0.001). PCR analysis of inos (F) and tnf-α, (G) mRNA levels of control macrophages (Mac) and Mac with degenerated discs (IL-1 stimulation) (*p < 0.05); H. Nitrite contents in culture media using the Griess assay. The polarized macrophage morphology after co-culture with IL-1 treated discs in shown in SI: Fig. S9D.

Fig. 5.

Macrophage activation by degenerated discs is reversed by curcumin treatment of discs by comparing control macrophages (Mac), Mac with discs pre-treated with IL-1β 10 ng/mL for 1 day (Mac+(IL-1)disc), and Mac with discs pre-treated with IL-1β 10 ng/mL and curcumin 10 μM for 1 day (Mac+(IL-1+Cur)disc) based on: A. Scatter plot (and histograms) of φZ_{2 MHz} vs. φZ_{18 MHz} show downshifting with degenerated discs and its reversal in presence of curcumin. B. Histograms of electrical diameter; Activation reversal: C. Not significant based on mean electrical diameter; but indicated by images D(i)-D(iii) and associated image analysis in Supplementary Information (Fig. S10); E. Not significant based on mean φZ_{2 MHz}; F. Significant based on mean φZ_{18 MHz} differences between samples (*p < 0.05 level, **p < 0.01). G. Nitrite secreted in cell culture supernatant measured using the Griess assay indicates reversal of the activation phenotype (*p < 0.05), albeit not at single-cell sensitivity. Cross-validation based on mRNA expression is shown in SI: Fig. S8.

Fig. 6.

Macrophage activation at varying doses of IL-1 pretreatment and curcumin treatment to reverse the phenotype is benchmarked versus that obtained after stimulation with varying doses and duration levels of LPS, using the metrics of: A. φZ_{2 MHz}, B. φZ_{18 MHz}, and C. electrical diameter or $\sqrt[3]{{\left|Z\right|}_{0.5\text{MHz}}}$ to highlight the dynamic range of the sensor for quantitative comparisons (*p < 0.05 level, **p < 0.01, ***p < 0.001). The respective significance levels after LPS treatment are in Fig. 3.