IL-1Ra deficiency accelerates intervertebral disc degeneration in C57BL6J mice

Abstract

The expression of Interleukin-1ß (IL-1ß) and its antagonist and Interleukin-1 receptor antagonist (IL-1Ra) are correlated with greater human intervertebral disc (IVD) degeneration, suggesting that elevated IL-1β activity promotes disc degeneration. Many in vitro studies support such a mechanistic relationship, whereas few in vivo investigations have been reported. The present study tests the effect of increased IL-1β activity on intervertebral disc in mice with an IL-1Ra gene deletion. IL-1Ra-/- mice and wild-type (WT) C57Bl6J mice were examined at 3 and 12 months of age. Caudal IVD segments were evaluated for disc degeneration by histopathology, functional testing, and inflammatory gene expression relevant to IL-1β pathways. To test differences in injury response, pinprick annular puncture was performed on IL-1Ra-/- and WT mice and evaluated similarly. IL-1Ra-/- IVDs had significantly worse histopathology at 3 months compared to WT controls, but not at 12 months. IL-1Ra-/- IVDs exhibited significantly more viscous mechanical properties than WT IVDs. qPCR revealed downregulation of inflammatory genes at 3 and 12 months in IL-1Ra-/- IVDs, with concomitant downregulation of anabolic and catabolic genes. Annular puncture yielded no appreciable differences between 2-week and 6-week post-injured WT and IL1-Ra-/- IVDs in histopathology or biomechanics, but inflammatory gene expression was sharply downregulated in IL-1Ra-/- mice at 2 weeks, returning by 6 weeks post injury. In the present study, IL-1Ra deletion resulted in increased IVD histopathology, inferior biomechanics, and transiently decreased pro-inflammatory cytokine gene expression. The histopathology of IL-1Ra-/- IVDs on a C57BL/6J background is less severe than a previous report of IL1Ra-/- on a BALB/c background, yet both strains exhibit IVD degeneration, reinforcing a mechanistic role of IL-1β signaling in IVD pathobiology. Despite a pro-inflammatory environment, the annular puncture was no worse in IL-1Ra-/- mice, suggesting that response to injury involves pathways other than inflammation. Overall, this study supports the hypothesis that IL-1β-driven inflammation is important in IVD degeneration.

Keywords: IL‐1ra deficiency; intervertebral disc; mouse strain.

FIGURE 1

Uninjured IVDs stained with H&E. (A) 3‐month wild‐type mice mid‐sagittal histological images in 3‐month mid‐sagittal images, a consistent pattern is seen, with a central strip of flattened notochordal cells (black arrows). From the central strip, finger‐like extensions of notochordal cells (pink arrows) seem to extend radially. IL‐1Ra−/− NP regions exhibit altered cellularity with partial loss of notochordal cells. In addition, the central strip is larger, and the finger‐like extensions are less in number and cells. (B) 12‐Month Wild‐Type Mid‐Sagittal Images. At 12 months, we see an expansion of the central strip seen in 3‐month samples. Vacuolated notochordal cells surround the central area, which is filled with primarily smaller chondrocyte cells. In 12‐month mid‐sagittal images, the central strip is much narrower and contains areas of cellular debris. In addition, an identifiable border develops between peripheral matrix and notochordal cells. (C) Dot plot with median values showing histological score frequencies between IL‐1Ra−/− groups (KO) and WT groups. While there were occasional outliers with highly degenerative scores of 9 in either genotype, most scores clustered toward the lower end of the scale, indicating relatively normal structure in knockout IVDs. However, IL‐1Ra−/− scores were significantly higher at 3 months as per Kruskal–Wallis test (p = 0.0003)

FIGURE 2

(A) Uninjured elastic biomechanical measures summary. No significant differences were seen between WT and IL‐1Ra−/− mice in most elastic measures. However, significant differences were seen between the IL‐1Ra−/− and WT mice at 3 months in NZ stiffness, but not in compressive or tensile stiffness (B) Uninjured Dynamic Mechanical Measures. As expected, phase angles were largest at the lower frequencies and smallest at the higher frequencies, in all groups. IL‐1Ra−/− mice had significantly higher phase angles at 0.05 and 0.1 Hz, with significant differences between 3‐month IL‐1Ra−/− vs WT, and also significant differences between 12‐month IL‐1Ra−/− vs WT. There were no differences in high‐frequency loading conditions (1 Hz and 10 Hz). Consequently, loss stiffness and tan delta were also significantly different between IL‐1Ra−/− and WT mice at lower frequency loading. Storage stiffness was different at all frequencies

FIGURE 3

L‐1Ra−/− inflammatory, matrix anabolic and proteolytic gene expression from caudal IVDs. All inflammatory genes showed a statistically significant difference via ANOVA, especially between the 3‐month anchor and other groups. Inflammatory gene expression for IL‐1β, TNF‐α, and NF‐kB in 3‐ and 12‐month groups was much less in IL‐1Ra−/− mice compared to the 3‐month wild‐type anchor. For matrix anabolic genes, there were no differences at 3 months, while collagen II gene expression was significantly higher in IL‐1Ra ‐/‐ mice at 12 months than WT mice. For matrix proteolytic genes, MMP‐3 and ADAMTS‐5 gene expressions were lower in all groups when compared to the WT 3‐month group

FIGURE 4

(A) Immunohistochemistry of IL‐1β and matrix proteins in uninjured caudal IVDs. IVDs underwent immunohistochemical staining with anti‐IL‐1β antibody and biotinylated secondary antibody, with HRP‐DAB (dark brown color) visualization. Intense IL‐1β staining was seen in the NP in most samples, with no difference between WT and IL‐1Ra−/−. (B) Immunohistochemistry of matrix anabolic proteins (Col2 and ACAN) and catabolic enzymes (MMP‐3 and ADAMTS‐5). Despite some differences in distribution between NP and AF for MMP‐3 (more in NP) and ADAMTS‐5 (more in AF), there were no differences between groups, even though there were gene expression differences

FIGURE 5

Injured caudal IVDs stained with H&E. Injured IVDs at 2 weeks (A) had significant annular morphological differences as compared to uninjured IVDs, but there were no significant differences between groups. At 6 weeks post injury (B), there was significant variation in magnitude of injury, but there were no differences between groups

FIGURE 6

(A) Injured caudal IVD elastic biomechanical properties were not different between groups at 2 or 6 weeks post injury. (B) Dynamic mechanical properties were also not different between groups

FIGURE 7

Inflammatory gene expression of injured wild‐type and IL‐1Ra−/− caudal IVDs at 2 and 6 weeks post injury. At 2 weeks post injury, there was significant upregulation of IL‐1β, TNF‐α, and eotaxin in WT mice, while there was significant downregulation of the same genes in IL‐1Ra−/− mice. At 6 weeks post injury, there was significant downregulation in both WT and IL‐1Ra−/− groups, as compared to the baseline condition of 3‐month uninjured WT mice; however, there was no difference between the WT and IL‐Ra−/− mice at 6 weeks post injury