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 use single cell RNA-sequencing analysis of cells extracted from the 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 two- and eight-week post-operative time points. The cells from these IVDs were extracted and transcriptionally profiled at a single-cell resolution. Unsupervised cluster analysis revealed the presence of 4 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 2- and 8- 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.

Identification of Cervical Disc Degeneration and Genes Associated with Immune Infiltration

“…Chemokines released from degenerated IVD promote the in ltration and activation of immune cells such as T lymphocytes, B lymphocytes, macrophages, and mast cells, which can trigger a series of pathological responses in IVD cells, including promoting autophagy, aging, or apoptosis. On the other hand, it further ampli es the in ammatory cascade reaction and promotes the release of neurotrophic proteins, especially NGF, inducing nerve ber growth into the IVD, causing discogenic pain [17] . Previous studies have shown that immune cell in ltration (including Tregs and macrophages) is involved in the progression of IDD [18,19] .…”