Oral Presentation 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

INTRODUCTION

Modic type 1 changes (MC1) are a source of nociceptive pain. Disease modifying treatments are missing because the underlying pathophysiology remains largely unknown. Pathomechanisms may include aspects of bacterial defense response and autoimmune response to disc matrix constituents. However, it remains unknown which cell types and immune mechanisms are triggered in bone marrow of MC1. Understanding these mechanisms is critical to define targeted interventions. Therefore, the aim of this project was to immunophenotyping the bone marrow of MC1.

METHODS

Vertebral bone marrow aspirates (n=8 MC1 + 8 intra-patient controls) were obtained from spinal fusion patients. Mononuclear cells were analyzed by a 36-color spectral flow cytometry-based immunophenotyping panel combined with 360 PE-labeled antibodies against surface markers. To avoid batch effects, each sample was barcoded with a unique combination of fluorescently labeled anti-CD45 antibodies, and all samples were analyzed in one tube. Data was computationally demultiplexed, gated into immune cell populations, and the co-expression of screened markers on the surface of these populations was analyzed with Infinity-flow algorithm.

To validate findings vertebral bone marrow aspirates from four additional MC1 patients undergoing lumbar spinal fusion were analyzed with single-cell RNA-sequencing (scRNAseq) (n=4 MC1 + 4 intra-patient controls). Single cell suspensions were created by a combination of enzymatic and mechanical release. 10,000 monocuclear cells (CD45+CD66b-) per sample were sequenced. Cell fractions were calculated on curated data and compared between MC1 and intra-patient control with paired t-tests. Within clusters of interest, pseudobulk differential expression analysis and overrepresentation analysis between MC1 and intra-patient controls was performed.

RESULTS

Plasmocytoid dendritic cells (pDC) were among the top enriched subsets in MC1 compared to intra-patient controls in both cohorts (flow:+19%, scRNAseq:+55%, both fdr<0.001) (Fig. 1A, 2A). With flow cytometry, cytotoxic Vδ1 T cells (+15%, fdr<0.001) were found upregulated. With scRNAseq CD8 T-cells, the umbrella subset of Vδ1, was also upregulated (+32%, fdr<0.001). Of all T cells, Vδ1 cells expressed most of the activating receptor NKP80 (Fig. 1B), suggesting that recognition of stress-induced molecules could trigger accumulation of Vδ1 cells in MC1. NKP80-expressing Vδ1 cells were the most cytotoxic T cell subset (Fig. 1C), were enriched in MC1 bone marrow (Fig. 1D), and overexpressed TLR4 (log2fc=1.9, fdr<0.001). pDC also overexpressed TLR4 (log2fc=0.4, fdr<0.05) (Fig.2B) and with “Adaptive immune response” as the top enriched pathway (Fig. 2C), indicating that pDC may bridge innate and adaptive immune response in MC1 (Fig.3).

DISCUSSION

Accumulation of pDCs and cytotoxic T cells with overexpression of TLR4 in both cell subset was consistently found in MC1 using two complementary approaches on two independent cohorts. This indicates immune system activation in MC1, possibly through TLR4. Vδ1 and pDC are both cell types bridging innate and adaptive immune system and have been associated with autoimmune diseases and chronic inflammation. Understanding the role of these immune cell subsets in MC1 might represent a key step toward designing targeted treatments for MC1.