Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

INTRODUCTION: Intervertebral disc (IVD) degeneration (IDD) is a multifactorial condition that progresses with age. Regardless of the cause, inflammation is an omnipresent contributor to IDD. The IVD is a composite connective tissue structure: central nucleus pulposus (NP), encapsulating annulus fibrosus (AF), and cartilage endplate. Importantly, age-associated changes in IVD tissue types, and the relationship between the effects of aging and inflammation on IDD remains unknown. The objective of the study was to use the next generation sequencing (bulk RNA sequencing, and single-cell RNA sequencing) to assess transcriptomic profiles in the IVD tissue types and immune cell population in aging mice.

METHODS: Bulk RNA Sequencing: NP and AF tissues from lumbar IVDs were dissected from C57BL/6 mice at 6, 12, 16, and 20+ months-of-age (M= months-of-age; n=8 for 6M, 12M, 16M; n=2 for 20+M). For each animal, IVD tissue types were pooled, total RNA was extracted, library was prepared (STRPOLYA kit), and sequenced (Illumina NovaSeq6000). Gene count data were normalized using DESeq2. Differentially expressed genes (DEGs) were identified based on |log2FoldChange| ≥ 1.5 and adjusted p value (padj.) < 0.05. Analyses were performed in R to identify differences within NP or AF tissues at various age compared to 6M. Functional enrichment analysis was performed using gene set enrichment analysis (GSEA) in clusterProfiler package. Single-Cell RNA Sequencing (scRNA-Seq): L4-S1 lumbar IVDs dissected from C57BL/6 mice at ages listed above (n=2/age) were sequentially digested for 2 hr. Viable cells were FAC sorted and scRNA-Seq was performed using 10x Genomics Chromium. Following data alignment and conversion, cells with <15% mitochondrial features and gene counts > 200 were analyzed. Principal component analysis and uniform manifold approximation and projection (UMAP) were performed to identify cell populations, and cluster identity were assigned using STRING database. All analyses and data visualization were performed in R (Seurat).  

RESULTS: Bulk RNA Sequencing: In both NP and AF tissues, commonly upregulated genes included immunoglobulins and cell surface proteins associated with B cell activation (Ifitm1 in NP, Fcrl5 in AF; Fig. 1.A,B). In both NP and AF, GSEA showed B cell activation and phagocytosis recognition as enriched biological processes in all age groups, suggesting potential role of macrophages and B cells in IVD aging (Fig. 1.C,D). scRNA-Seq: Clustering analysis of 36,480 cells resolved 9 unique clusters, whose identity (Neutrophil, B Cells 1& 2, Macrophage, Monocyte, T Cell, IVD Cells and Granulocytes) was defined based on transcriptome profile (Fig.1.E). Longitudinal cluster cell count showed age-dependent increase in B Cell-1 and macrophage clusters (Fig.1.F). Sub-clustering analysis of B Cell-1 revealed age-dependent increase in activated B cell markers (Fcmr, Ms4a1, Cd83; Fig.1.G). Macrophage cluster showed increased expression of M1 macrophage markers (Cd38, Serpinb2, Tlr4) and low expression of M2 markers (Mrc1, Arg1, Il10; Fig.1.H).

DISCUSSION: Our findings suggest that NP and AF tissues upregulate genes associated with B cell activation with age. Given that our analysis showed increased M1 macrophage marker expression with age, the crosstalk between IVD, M1 macrophages, and B cells may be potential immune pathways contributing to the IDD.