Inflammation induces irreversible biophysical changes in isolated nucleus pulposus cells

Abstract

Intervertebral disc degeneration is accompanied by elevated levels of inflammatory cytokines that have been implicated in disease etiology and matrix degradation. While the effects of inflammatory stimulation on disc cell metabolism have been well-studied, their effects on cell biophysical properties have not been investigated. The hypothesis of this study is that inflammatory stimulation alters the biomechanical properties of isolated disc cells and volume responses to step osmotic loading. Cells from the nucleus pulposus (NP) of bovine discs were isolated and treated with either lipopolysaccharide (LPS), an inflammatory ligand, or with the recombinant cytokine TNF-α for 24 hours. We measured cellular volume regulation responses to osmotic loading either immediately after stimulation or after a 1 week recovery period from the inflammatory stimuli. Cells from each group were tested under step osmotic loading and the transient volume-response was captured via time-lapse microscopy. Volume-responses were analyzed using mixture theory framework to investigate two biomechanical properties of the cell, the intracellular water content and the hydraulic permeability. Intracellular water content did not vary between treatment groups, but hydraulic permeability increased significantly with inflammatory treatment. In the 1 week recovery group, hydraulic permeability remained elevated relative to the untreated recovery control. Cell radius was also significantly increased both after 24 hours of treatment and after 1 week recovery. A significant linear correlation was observed between hydraulic permeability and cell radius in untreated cells at 24 hours and at 1-week recovery, though not in the inflammatory stimulated groups at either time point. This loss of correlation between cell size and hydraulic permeability suggests that regulation of volume change is disrupted irreversibly due to inflammatory stimulation. Inflammatory treated cells exhibited altered F-actin cytoskeleton expression relative to untreated cells. We also found a significant decrease in the expression of aquaporin-1, the predominant water channel in disc NP cells, with inflammatory stimulation. To our knowledge, this is the first study providing evidence that inflammatory stimulation directly alters the mechanobiology of NP cells. The cellular biophysical changes observed in this study are coincident with documented changes in the extracellular matrix induced by inflammation, and may be important in disease etiology.

Figure 1. Effects of LPS and TNF-α on inflammatory expression of NP cells.

(A) Total nitrite release at days 0, 1, 4, and 7 post stimulation. *p<0.001 relative to the immediately preceding time point. (B) IL-1β gene expression level measured after 24 hours (untreated, LPS, TNF-α) and (C) after 1 week recovery period (untreated recovery, LPS recovery, TNF-α recovery). IL-1β gene expression levels in B and C are normalized to untreated or untreated recovery groups, respectively. ⁺p<0.001 vs. untreated or untreated recovery group.

Figure 2. Cell radius measured at 333 mOsm/L at (A) 24 hour and (B) 7-day recovery time points.

Data shown is mean (▪), standard error (boxes), and 95% confidence intervals (whiskers; n = 52–127 cells per group). *p<0.03 vs. untreated or untreated recovery control.

Figure 3. Osmotic loading regime, representative volume-response curve, and equilibrium volume-concentration responses.

(A) Osmolarity was applied in a step-wise fashion using decreasing concentrations of NaCl solutions. Each osmotic load was maintained for approximately 5 minutes to allow the cell volume change to reach equilibrium. (B) Representative time-dependent volume of an untreated cell. (C, D) Equilibrium volume (V), normalized by reference volume (V_r), is plotted as a function of applied external reference osmolarity (c_er) over current external osmolarity (c_e) for cells at (C) 24 hour and (D) 7-day recovery time points (n = 7–12 cells per group). The reference osmolarity and associated volume were taken at 333 mOsm/L equilibrium.

Figure 4. Mean intracellular water content (A, B) and mean hydraulic permeability (C, D) at 333 mOsm/L step for cells at (A, C) 24 hour and (B, D) 7-day recovery time points, respectively.

*p<0.05 vs. untreated or untreated recovery control. n = 7–12 cells per condition.

Figure 5. Regression analysis between and cell radius in each treatment group.

Significant linear correlations were observed for cells in (A) untreated and (D) untreated recovery groups (p<0.05, indicated in red). No significant correlations were observed for cells from LPS or TNF-α at 24 hours (B, C) or at recovery time points (E, F). ‘R’ represents the regression coefficient for each group.

Figure 6. Aquaporin-1 expression in NP cells.

(A, B) Gene expression of aquaporin-1 at 24 hour or 7-day recovery time points. Expression levels are normalized to untreated time point control groups (*p<0.05 vs. control). (C) Representative immunoblot of Aqp-1 and (D, E) densitometry of Aqp-1 in NP cells at 24 hour or 7-day recovery time points, respectively. Expression levels are normalized to untreated time point control groups. *p<0.05 vs. time point control, ∧p = 0.07 vs. time point control. (F) Representative immunohistochemical (IHC) staining of Aqp-1 (green) and cell nuclei (blue, scale bars = 20 µm). (G, H) Quantification of Aqp-1 expression measured by IHC, normalized to time point untreated control group (*p<0.05 vs. time point control).

Figure 7. Cytoskeletal structure of rounded NP cells.

Representative cytoskeletal staining of NP cells in each treatment group for (A–F) F-actin (red) and (G–L) β-tubulin (green), scale bar = 5 µm. Confocal z-stack images were acquired with 2 µm spacing throughout cell cross-sections, and images represent the slice acquired nearest the center of the cell (midplane).