Single Cell RNA-Sequence Analyses Reveal Uniquely Expressed Genes and Heterogeneous Immune Cell Involvement in the Rat Model of Intervertebral Disc Degeneration

Abstract

Intervertebral disc (IVD) degeneration is characterized by a loss of cellularity, and changes in cell-mediated activity that drives anatomic changes to IVD structure. In this study, we used single-cell RNA-sequencing analysis of degenerating tissues of the rat IVD following lumbar disc puncture. Two control, uninjured IVDs (L2-3, L3-4) and two degenerated, injured IVDs (L4-5, L5-6) from each animal were examined either at the two- or eight-week post-operative time points. The cells from these IVDs were extracted and transcriptionally profiled at the single-cell resolution. Unsupervised cluster analysis revealed the presence of four known cell types in both non-degenerative and degenerated IVDs based on previously established gene markers: IVD cells, endothelial cells, myeloid cells, and lymphoid cells. As a majority of cells were associated with the IVD cell cluster, sub-clustering was used to further identify the cell populations of the nucleus pulposus, inner and outer annulus fibrosus. The most notable difference between control and degenerated IVDs was the increase of myeloid and lymphoid cells in degenerated samples at two- and eight-weeks post-surgery. Differential gene expression analysis revealed multiple distinct cell types from the myeloid and lymphoid lineages, most notably macrophages and B lymphocytes, and demonstrated a high degree of immune specificity during degeneration. In addition to the heterogenous infiltrating immune cell populations in the degenerating IVD, the increased number of cells in the AF sub-cluster expressing Ngf and Ngfr, encoding for p75NTR, suggest that NGF signaling may be one of the key mediators of the IVD crosstalk between immune and neuronal cell populations. These findings provide the basis for future work to understand the involvement of select subsets of non-resident cells in IVD degeneration.

TRPV4 differentially controls inflammatory cytokine networks during static and dynamic compression of the intervertebral disc

“…The reduction of these T‐cell recruiting chemokines on Day 7 indicates TRPV4 activation may be protective against the infiltration of delayed T‐cells into the IVD. It has been shown that at 2 weeks post‐injury in a rat model of IVD degeneration and LBP, immune cells, including T‐cells, are significantly increased 62 . Based on our findings here, there is potential to mediate this immune cell infiltration using controlled TRPV4 activation.…”

“…It has been shown that at 2 weeks post-injury in a rat model of IVD degeneration and LBP, immune cells, including T-cells, are significantly increased. 62 Based on our findings here, there is potential to mediate this immune…”

Analysis of Infiltrating Immune Cells Following Intervertebral Disc Injury Reveals Recruitment of Gamma-Delta (γδ) T cells in Female Mice

“…Human patient discectomy samples have been shown to contain Cd4+ T cells, but these tissues are in a chronic state of IVD degeneration and do not give insight into the role the T cells play in the acute healing stages 43,45 . Rodent models have shown the presence of Cd4+ or Cd8+ T cells at isolated time points, but rarely address sex-related differences or subtype differences 45–47 . We discovered drastic differences in the responding lymphocytes between the sexes at 3 dpi where DN T cells dominated the female response but Cd8+ and NK T cells were responders in males with injury ( Figure 4 ).…”

Intervertebral disc degeneration—Current therapeutic options and challenges

“…Also, non-lethal polymorphisms in early IVD patterning genes will likely surface over time as underlying cause. Furthermore, both single cell and bulk transcript analysis of IVD derived cells through RNASeq and other methods will likely point to biomarkers for NP and AF cells of healthy or degenerated discs worth investigating in IVDD linkage analysis (41,42,(194)(195)(196)(197)(198). Going forward it will be crucial to investigate not only polymorphisms in coding and regulatory regions such as promoters or enhancer/silencer binding sites of genes but also epigenetic modifications from methylation, acetylation and lactylation involved in metabolic reprogramming among other effects (197,199).…”