The cGAS-STING pathway affects vertebral bone but does not promote intervertebral disc cell senescence or degeneration

Abstract

The DNA-sensing cGAS-STING pathway promotes the senescence-associated secretory phenotype (SASP) and mediates type-I interferon inflammatory responses to foreign viral and bacterial DNA as well as self-DNA. Studies of the intervertebral disc in humans and mice demonstrate associations between aging, increased cell senescence, and disc degeneration. Herein we assessed the role of STING in SASP promotion in STING gain- (N153S) and loss-of-function mouse models. N153S mice evidenced elevated circulating levels of proinflammatory markers including IL-1b, IL-6, and TNF-a and exhibited a mild trabecular and cortical bone phenotype in caudal vertebrae. Interestingly, despite systemic inflammation, the structural integrity of the disc and knee articular joint remained intact, and cells did not show a loss of their phenotype or elevated SASP. Transcriptomic analysis of N153S tissues demonstrated an upregulated immune response by disc cells, which did not closely resemble inflammatory changes in human tissues. Interestingly, STING-/- mice also showed a mild vertebral bone phenotype, but the absence of STING did not improve the age-associated disc phenotype or reduce the abundance of SASP markers. Overall, the analyses of N153S and STING-/- mice that the cGAS-STING pathway is not a major contributor to SASP induction and consequent disc aging and degeneration but may play a minor role in the maintenance of trabecular bone in the vertebrae. This work contributes to a growing body of work demonstrating that systemic inflammation is not a key driver of disc degeneration.

SDC4 deletion perturbs intervertebral disc matrix homeostasis and promotes early osteopenia in the aging spine

“…A 0.4 N compressive preload was applied, followed by a monotonic displacement ramp at 0.1 mm/s until failure. Force-displacement data were digitally captured at 25 Hz and converted to stress-strain using a custom GNU Octave script with µCT-based geometric measurements, as previously described (55,56).…”

“…Following Safranin-O/Fast Green/Hematoxylin staining, images were acquired on a light microscope (Axio Imager 2; Carl Zeiss Microscopy) using 5x/0.15 N-Achroplan (Carl Zeiss) objective and Zen2™ software (Carl Zeiss). The health of disc compartments was assessed by at least four blinded graders using Modified Thompson Grading (56,(58)(59)(60)(61). Picrosirius red staining (Polysciences, 24901) was performed to assess collagen fibril thickness, and images were acquired using 4x /0.25 Pol /WD 7.0 (Nikon) objective on a polarizing light microscope (Eclipse LV100 POL; Nikon).…”

“…Picrosirius red staining (Polysciences, 24901) was performed to assess collagen fibril thickness, and images were acquired using 4x /0.25 Pol /WD 7.0 (Nikon) objective on a polarizing light microscope (Eclipse LV100 POL; Nikon). NIS Elements Viewer software (Nikon) was used to set color threshold for the level of green (thin), yellow (intermediate), and red (thick) fibers (56,60). Color threshold levels remained constant for all samples.…”

“…Imaging Fourier-Transform Infrared Spectroscopy 7 µm deparaffinized sections of decalcified lumbar disc tissues (L5/S1) were used to acquire infrared (IR) spectral imaging data using methods previously described (56,60). Spatial resolution images were acquired on the Spectrum Spotlight 400 FT-IR Imaging system (Perkin Elmer, Waltham, MA, USA) in the mid-IR (MIR) region from 4000 cm −1 to 740 cm −1 wavenumbers spectral range, at 25 µm pixel resolution, 8 scans per pixel, and a spectral resolution of 8 cm -1 .…”

“…Raw spectra went through an atmospheric correction and converted to second derivative spectra to resolve underlying peaks and corrected for baseline offsets. Savitsky-Golay smoothing (filter order of 3 and filter length of 9) was applied, and spectra inverted by multiplication by -1 to convert to positive peak heights as appropriate (56,60). Mean second-derivative absorbances in the amide I (1660 cm -1 ), amide II (1549 cm -1 ), collagen side chain vibration (1338 cm -1 ), and sulfated proteoglycan sugar ring (1064 cm -1 ) peaks were quantified and compared in the NP and AF compartments.…”