Dynamic imaging demonstrates that pulsed electromagnetic fields (PEMF) suppress IL-6 transcription in bovine nucleus pulposus cells

Abstract

Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Pulsed electromagnetic fields (PEMF) are noninvasive biophysical stimulus that has been used extensively in the orthopaedic field for many years. However, the specific cellular responses and mechanisms involved are still unclear. The objective of this study was to assess the time-dependent PEMF effects on pro-inflammatory factor IL-6 expression in disc nucleus pulposus cells using a novel green fluorescence protein (GFP) reporter system. An MS2-tagged GFP reporter system driven by IL-6 promoter was constructed to visualize PEMF treatment effect on IL-6 transcription in single living cells. IL-6-MS2 reporter-labeled cells were treated with IL-1α to mimic the in situ inflammatory environment of degenerative disc while simultaneously exposed to PEMF continuously for 4 h. Time-lapse imaging was recorded using a confocal microscope to track dynamic IL-6 transcription activity that was demonstrated by GFP. Finally, real-time RT-PCR was performed to confirm the imaging data. Live cell imaging demonstrated that pro-inflammatory factor IL-1α significantly promoted IL-6 transcription over time as compared with DMEM basal medium condition. Imaging and PCR data demonstrated that the inductive effect of IL-1α on IL-6 expression could be significantly inhibited by PEMF treatment in a time-dependent manner (early as 2 h of stimulus initiation). Our data suggest that PEMF may have a role in the clinical management of patients with chronic low back pain. Furthermore, this study shows that the MS2-tagged GFP reporter system is a useful tool for visualizing the dynamic events of mechanobiology in musculoskeletal research. © 2017 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:778-787, 2018.

Keywords: IL-6 mRNA expression; MS2-GFP reporter; dynamic imaging; pulsed electromagnetic fields; spine/disc biology.

Figure 1

Schematic of MS2 constructs used in this study. Plasmid A) PGK‐MS2‐GFP fusion protein Plasmid, expresses the MS2‐GFP fusion protein; Plasmid B) reporter mRNA, expresses an MS2 × 24 repeat sequence driven by IL‐6 promoter (IL‐6‐RFP‐MS2). Single transfected cells (Plasmid A, left side) showed a diffuse green fluorescence concentrated within the cell nucleus because of the nuclear localization sequence (NLS); In co‐transfected cells (right side), the fusion protein co‐expresses with the reporter mRNA containing MS2‐binding sites. The fusion protein specifically binds to one of the 24 copies of the RNA motif contained in the MS2 × 24 repeat sequence. The resultant binding of multiple copies of the MS2‐GFP fusion protein to the mRNA forms bright green particles in cells (particularly induced by pro‐inflammatory factor IL‐1α).

Figure 2

Visualization of dynamic mRNA expression in individual live cells. A1) Illustration of IL‐6 expression ON and OFF in MS2‐GFP system. A2) Single GFP particles in living cells after transfection. B) GFP particle distribution in single living cells after co‐transfection (both in the cytoplasm and nucleus); White dots represent GFP particles that are tracked with Imaris software. C) Dynamic imaging of GFP in nucleus of living cells after co‐transfection (induced by IL‐1α) for 400 min. Exposure time is reduced to visualize the particulate nature of the intense nuclear stain.

Figure 3

Flow cytometry verifying the GFP and RFP profile in MS2‐GFP fusion and mRNA reporter system. A) Control, without transfection. B) MS2‐GFP fusion plasmid single transfection C) IL‐6‐RFP‐MS2 plasmid single transfection D) Co‐transfection (n = 3/group). MFI = mean fluorescence intensity.

Figure 4

Dynamic images demonstrating the effects of IL‐1α alone and IL‐α + PEMF treatment on IL‐6 expression. PEMF dosing started at 2 h after IL‐1α treatment.

Figure 5

Quantification of IL‐1α and PEMF treatment effects by calculating GFP particle number over time (n = 9/group). PEMF dosing started at 2 h after IL‐1α treatment (blue arrow) and turned off at 6 h (red arrow). Significant differences between IL‐1α and IL‐1α + PEMF group starts at 2hr (p < 0.01). Loss of significance occurred between IL‐α + PEMF and DMEM groups at 3.5 h (p < 0.05).

Figure 6

Real time RT‐PCR validation for PEMF and IL‐1 α treatment effects. # represents p = 0.07; ** represents p < 0.01. n = 3 for biological replicates and n = 3 technical replicates.