Integrating multiple omics analyses to elucidate the macrophage-driven “immune cellular senescence fibrosis” axis mechanism in intervertebral disc degeneration

The primary pathological process responsible for low back pain is intervertebral disc degeneration (IVDD), which is typified by an imbalance in the immunological microenvironment and fibrotic phenotypic alterations of nucleus pulposus cells (NPCs). The aberrant deposition of extracellular matrix, such as collagen, is a feature of NPC fibrosis, and the dysregulation of the immune milieu is intimately linked to the aberrant polarization of macrophages, both of which contribute to the development of IVDD1. Thus, investigating the inherent relationship between NPC fibrosis and alterations in the immune microenvironment is crucial for understanding the pathophysiology of IVDD. This approach seeks to methodically identify the intervertebral disc fibrosis repair mechanism, offering a fresh theoretical foundation for developing targeted treatments to slow or stop the disease’s advancement2–4. The study follows the Transparency in Artificial Intelligence Reporting – TITAN guidelines5. A total of 1751 differentially expressed genes were found, of which 29 were upregulated, and 1722 were downregulated (Supplemental Digital Content Table S1, available at: https://links.lww.com/JS9/G595). Fifty-eight important genes were found. Ten hub genes were chosen using protein interaction network analysis. Expression validation revealed statistical differences across groups for EGFR, MTOR, CREB1, SMAD2, CRP, and NLRP3 (Supplemental Digital Content Figures 1 and 2, available at: https://links.lww.com/JS9/G594). After strict quality control, the cells were successfully divided into 11 subgroups through clustering and cell annotation. We classified the 0–3 and 10 cell subtypes as NPCs based on the classic markers of ANC and SOX9, and the 4–9 cell subtype as immune cells. NP cells were further refined and divided into 0 hypertrophic chondrocyte-like NP cells (HT-CLNPs), 1 fibro-NP cells (fibro-NPCs), 2 adhesion NP cells (adhesion-NPCs), 3 regulatory NP cells (regulatory-NPCs), and 10 homeostatic NP cells (homeostatic-NPCs). Immune cells are further divided into Myelocyte, Macrophage, T cells, Endothelial cells, NK cell, and B cells. From the results, it can be seen that immune cells mainly exist in mildly degenerated NP tissues, while fibro-NPCs, adhesion-NPCs, regulatory-NPCs, and homeostatic–NPCs are mainly distributed in severely degenerated NP tissues. HT-CLNPs are expressed in both mild and severe NP tissues (Fig. 1C). Cell communication in NP samples with varying stages of degeneration was examined using CellChat. The addition of immune cells to mildly degenerated tissue suggests that immune cells infiltrated NP in the early stages of degeneration and changed the cell communication in the original NP (Fig. 1D). The figure shows that intercellular communication in severely degenerated NP tissue is significantly increased compared to mildly degenerated tissue. The signaling pathways are mainly concentrated in HT-CLNPs, fibro-NPCs, adhesion-NPCs, and homeostatic-NPCs. Therefore, we preliminarily infer that with the addition of immune cells such as macrophages and NK cells in degenerated NP tissue, the MIF signaling pathway network is initiated, and NP cells gradually undergo pathological progression such as adhesion, hypertrophy, and fibrosis. Figure 1.: (A) Schematic diagram of inflammatory factors, immune cells, especially macrophages, infiltration patterns during normal and degenerated intervertebral discs. (B) Molecular mechanism diagram of macrophage-driven intervertebral disc degeneration through “immune cell aging fibrosis.” (C) A myeloid cell atlas was constructed based on single-cell analysis, mainly including some immune cells and degenerated myeloid cells, such as macrophages, T cells, and fibrotic myeloid cells. (D) Cell-to-cell communication based on single cells. (E) Key proteins and signaling pathway mechanisms of cellular aging. (F) TGF-β signaling pathway mechanisms and key proteins. Perform quasi-temporal analysis on immune cells (macrophages, T cells, M2 macrophages, B cells) that play an important role in IVDD. Monocle2 trajectory analysis shows that macrophages are at the starting point of their developmental trajectory and subsequently affect the fate of B cells, M2 macrophages, and T cells, with the most significant difference in differentiation trajectory between M2 macrophages and T cells. CytoTRACE analysis further revealed the specific expression patterns of key immune marker genes in these immune cell subpopulations. For example, NFKB1 is mainly expressed in T cells, M2 macrophages, and B cells; IL1B, NLRP3, CCL3, and IL18 are mainly enriched in B cells and M2 macrophages (Fig. 2A and B). Figure 2.: (A) A pseudo-temporal analysis, with black dots representing differentially expressed genes selected based on classification. The principal component cell clustering distribution t-SNE plot shows that different colors represent branches of different cell fates in cell populations. The t-SNE temporal plot of cell clustering distribution. (B) Trajectory analysis and expression heatmap of immune marker genes CytoTRACE based on cell populations. Five core intersecting genes – IL6, BCL2, EGFR, NLRP3, and CD36 – were obtained by intersecting the hub genes chosen by bulk RNA sequencing with single-cell immune cell marker genes. These five core genes’ important signaling pathways in IVDD were identified by KEGG enrichment analysis: Cellular processes include cell senescence, autophagy, and apoptosis. Environmental Information Processing: FoxO, NF-κB, PI3K–Akt, and HIF-1 signaling pathways. Organic Systems: Th17 cell development and the IL-17 signaling pathway (Supplemental Digital Content Figure 3A–C, available at: https://links.lww.com/JS9/G594). In conclusion, this study used multiomics joint analysis to identify important genes like EGFR, NLRP3, and IL6. It also showed that these genes work together to drive the degeneration of the intervertebral disc by controlling autophagy, cell aging, and signaling pathways like HIF-1, NF-κB, and IL-17 in the intricate interactions between NPCs and immune cells (particularly macrophages and T cells) (Fig. 1E and F). The current theory emphasizes that M1 macrophages and their pro-inflammatory factors indirectly participate in IVDD by inducing cellular aging. This study proposes a more breakthrough and direct “immune fibrosis axis”: M2-like macrophages directly initiate and maintain the fibrotic program of NPCs through the TGF-β/Smad pathway. This means that in the IVDD process, in addition to the classic “inflammation aging SASP” loop, there is also a parallel, direct, and efficient fibrotic pathway dominated by M2 macrophages. This explains why, in some cases, the degree of tissue fibrosis is not completely parallel to the overall level of inflammation and emphasizes that targeting M2 macrophages or their TGF-β signaling may be a more precise strategy for reversing fibrosis (Fig. 1A and B)6. The results of single-cell transcriptomics provide the basis of this investigation. The spatial adjacency link between M2 macrophages and fibroNPCs in tissue in situ has not yet been directly confirmed by spatial transcriptome data, despite the fact that computational biology techniques like CellChat have produced excellent predictions of intercellular relationships. In the meantime, direct confirmation of the causal chain we deduce is necessary due to the absence of functional trials or animal models. In light of the aforementioned constraints, we suggest a precise and detailed future research strategy: Validating the spatiotemporal dynamics of cell interactions in the tissue microenvironment using multiplex fluorescence immunostaining and spatial transcriptomics; building a co-culture system of macrophages and NPCs, and employ TGF-β pathway inhibitors for functional intervention to confirm that this signaling axis is the cause of fibrosis; Investigating the creation of a nanodrug delivery system that targets M2 macrophages in order to offer preclinical proof for future precise local therapy of intervertebral disc degeneration.