The Role of Intervertebral Disc Cartilage Catabolites in Modic Type 1 Changes

The thesis investigates the pathobiology of Modic changes (MCs), specifically focusing on Modic type 1 changes (MC1), emphasizing the complexity of the disease. Although MCs manifest as vertebral bone marrow lesions, the inflammation and degeneration observed in the neighboring intervertebral disc and cartilage endplate (CEP) are also closely linked to MCs. However, the underlying mechanisms and how the tissues crosstalk to induce MC development are unclear to this point. Yet, comprehending these pathobiological mechanisms is crucial for the development of targeted therapies, which are currently nonexistent. The thesis explores various aspects of MC1 addressing pathologic processes seen within the MC1 bone marrow, the CEP and the disc. Finally, the project's findings suggest a new disease mechanism for MC1 development initiated through MC1 specific disc degeneration. Chapter three investigated the impact of bone marrow stromal cells (BMSC) from MC1 on neurite outgrowth, using an in-vitro co-culture system. Multiple studies have reported an elevated presence of nerve fibers in the MC1 bone marrow and endplate, a definitive connection to the underlying cause has not been established. For the first time, the dysregulated MC1 BMSCs were directly linked to increased neurite outgrowth. With this knowledge, an important treatment target has been identified, paving the way for further studies to explore how to mitigate this effect. The fourth chapter explores the concept of a disc microbiome, particularly focusing on the MC1 and MC2 microbiomes, using metagenomic analysis. The disc microbiome challenges the conventional notion of disc sterility, necessitating clear methodological definitions for further investigation. Although our analysis confirmed the presence of a disc microbiome, the findings deviated from those of previous studies on degenerated MC discs, even after aligning the parameters of bioinformatic analysis to the previous studies. This study underscores the importance of not only standardizing bioinformatic analysis approaches but also further investigating factors that influence the bacterial composition of the disc microbiome. Differences could stem from sample preparation techniques or variations in the geographical and ethnic backgrounds of patients. Once clarified, the exploration of the disc microbiome will unlock numerous opportunities for diagnostic and treatment applications. The fifth chapter of the thesis highlights the to this point overlooked biological role of CEP cells. The experiments were able to confirm the presence of Toll-like receptors (TLRs) on CEP cells and particularly emphasized the presence of TLR2 as it was the only TLR that was upregulated through direct stimulation. The discovery of TLRs on CEP cells is noteworthy due to their high density compared to the cells in the adjacent disc and their ability to induce inflammation and promote the production of catabolic enzymes, potentially leading to endplate degeneration. This not only enhances our understanding of CEP cells but also reveals novel treatment targets. The sixth chapter presents the main project of the thesis, which focuses on understanding MC1 pathobiology and proposes a new mechanism for MC1 development beginning with MC1-specific disc degeneration. It is grounded in the fact that although disc degeneration is often observed in the disc adjacent to MC1, not all degenerated discs progress to MC1. The proposed MC1-specific disc degeneration is hypothesized to generate more fragments, triggering inflammation in the adjacent endplate, causing endplate destruction. This ultimately breaches the CEP barrier between the disc and bone marrow, allowing pro-inflammatory fragments (damage associated molecular patterns (DAMPs)) and inflammatory cytokines to spill over into the bone marrow, inducing MC1. Although the study is ongoing, significant discoveries have been made. MC1 discs were found to have a higher abundance of extracellular matrix derived fragments as well a greater abundance of the protease high temperature requirement serine protease 1 (HTRA1) than degenerated nonMC discs. In a subsequent phase, the abundant cartilage intermediate layer protein 1 (CILP1) fragments were successfully replicated by exposure to HTRA1 and were demonstrated to possess pro-inflammatory properties via TLR4 signaling activation, thereby classifying them as DAMPs. A CEP explant model was used to show that TLR activation can induce CEP tissue destruction, connecting the DAMP abundance in the disc to CEP damage. However, further experiments are necessary to address the gaps within the proposed mechanism. MC1 is a pathology associated with distinct inflammatory and pain-related processes, suggesting it could and should be treated specifically. This thesis demonstrates this and thus marks an important step towards targeted therapy for MC1

Bone marrow stromal cells in Modic type 1 changes promote neurite outgrowth

The pain in patients with Modic type 1 changes (MC1) is often due to vertebral body endplate pain, which is linked to abnormal neurite outgrowth in the vertebral body and adjacent endplate. The aim of this study was to understand the role of MC1 bone marrow stromal cells (BMSCs) in neurite outgrowth. BMSCs can produce neurotrophic factors, which have been shown to be pro-fibrotic in MC1, and expand in the perivascular space where sensory vertebral nerves are located. The study involved the exploration of the BMSC transcriptome in MC1, co-culture of MC1 BMSCs with the neuroblastoma cell line SH-SY5Y, analysis of supernatant cytokines, and analysis of gene expression changes in co-cultured SH-SY5Y. Transcriptomic analysis revealed upregulated brain-derived neurotrophic factor (BDNF) signaling-related pathways. Co-cultures of MC1 BMSCs with SH-SY5Y cells resulted in increased neurite sprouting compared to co-cultures with control BMSCs. The concentration of BDNF and other cytokines supporting neuron growth was increased in MC1 vs. control BMSC co-culture supernatants. Taken together, these findings show that MC1 BMSCs provide strong pro-neurotrophic cues to nearby neurons and could be a relevant disease-modifying treatment target

Intervertebral disc microbiome in Modic changes: Lack of result replication underscores the need for a consensus in low-biomass microbiome analysis

INTRODUCTION The emerging field of the disc microbiome challenges traditional views of disc sterility, which opens new avenues for novel clinical insights. However, the lack of methodological consensus in disc microbiome studies introduces discrepancies. The aims of this study were to (1) compare the disc microbiome of non-Modic (nonMC), Modic type 1 change (MC1), and MC2 discs to findings from prior disc microbiome studies, and (2) investigate if discrepancies to prior studies can be explained with bioinformatic variations. METHODS Sequencing of 16S rRNA in 70 discs (24 nonMC, 25 MC1, and 21 MC2) for microbiome profiling. The experimental setup included buffer contamination controls and was performed under aseptic conditions. Methodology and results were contrasted with previous disc microbiome studies. Critical bioinformatic steps that were different in our best-practice approach and previous disc microbiome studies (taxonomic lineage assignment, prevalence cut-off) were varied and their effect on results were compared. RESULTS There was limited overlap of results with a previous study on MC disc microbiome. No bacterial genera were shared using the same bioinformatic parameters. Taxonomic lineage assignment using "amplicon sequencing variants" was more sensitive and detected 48 genera compared to 22 with "operational taxonomic units" (previous study). Increasing filter cut-off from 4% to 50% (previous study) reduced genera from 48 to 4 genera. Despite these differences, both studies observed dysbiosis with an increased abundance of gram-negative bacteria in MC discs as well as a lower beta-diversity. Cutibacterium was persistently detected in all groups independent of the bioinformatic approach, emphasizing its prevalence. CONCLUSION There is dysbiosis in MC discs. Bioinformatic parameters impact results yet cannot explain the different findings from this and a previous study. Therefore, discrepancies are likely caused by different sample preparations or true biologic differences. Harmonized protocols are required to advance understanding of the disc microbiome and its clinical implications

FGF2 overrides key pro-fibrotic features of bone marrow stromal cells isolated from Modic type 1 change patients

Extensive extracellular matrix production and increased cell-matrix adhesion by bone marrow stromal cells (BMSCs) are hallmarks of fibrotic alterations in the vertebral bone marrow known as Modic type 1 changes (MC1). MC1 are associated with non-specific chronic low-back pain. To identify treatment targets for MC1, in vitro studies using patient BMSCs are important to reveal pathological mechanisms. For the culture of BMSCs, fibroblast growth factor 2 (FGF2) is widely used. However, FGF2 has been shown to suppress matrix synthesis in various stromal cell populations. The aim of the present study was to investigate whether FGF2 affected the in vitro study of the fibrotic pathomechanisms of MC1-derived BMSCs. Transcriptomic changes and changes in cell-matrix adhesion of MC1-derived BMSCs were compared to intra-patient control BMSCs in response to FGF2. RNA sequencing and quantitative real-time polymerase chain reaction revealed that pro-fibrotic genes and pathways were not detectable in MC1-derived BMSCs when cultured in the presence of FGF2. In addition, significantly increased cell-matrix adhesion of MC1-derived BMSCs was abolished in the presence of FGF2. In conclusion, the data demonstrated that FGF2 overrides key pro-fibrotic features of MC1 BMSCs in vitro. Usage of FGF2-supplemented media in studies of fibrotic mechanisms should be critically evaluated as it could override normally dominant biological and biophysical cues

Mutant SF3B1 promotes malignancy in PDAC

The splicing factor SF3B1 is recurrently mutated in various tumors, including pancreatic ductal adenocarcinoma (PDAC). The impact of the hotspot mutation SF3B1$^{K700E}$ on the PDAC pathogenesis, however, remains elusive. Here, we demonstrate that Sf3b1$^{K700E}$ alone is insufficient to induce malignant transformation of the murine pancreas, but that it increases aggressiveness of PDAC if it co-occurs with mutated KRAS and p53. We further show that Sf3b1$^{K700E}$ already plays a role during early stages of pancreatic tumor progression and reduces the expression of TGF-β1-responsive epithelial-mesenchymal transition (EMT) genes. Moreover, we found that SF3B1$^{K700E}$ confers resistance to TGF-β1-induced cell death in pancreatic organoids and cell lines, partly mediated through aberrant splicing of Map3k7. Overall, our findings demonstrate that SF3B1$^{K700E}$ acts as an oncogenic driver in PDAC, and suggest that it promotes the progression of early stage tumors by impeding the cellular response to tumor suppressive effects of TGF-β

Modic type 2 changes are fibroinflammatory changes with complement system involvement adjacent to degenerated vertebral endplates

Background Vertebral endplate signal intensity changes visualized by magnetic resonance imaging termed Modic changes (MC) are highly prevalent in low back pain patients. Interconvertibility between the three MC subtypes (MC1, MC2, MC3) suggests different pathological stages. Histologically, granulation tissue, fibrosis, and bone marrow edema are signs of inflammation in MC1 and MC2. However, different inflammatory infiltrates and amount of fatty marrow suggest distinct inflammatory processes in MC2. Aims The aims of this study were to investigate (i) the degree of bony (BEP) and cartilage endplate (CEP) degeneration in MC2, (ii) to identify inflammatory MC2 pathomechanisms, and (iii) to show that these marrow changes correlate with severity of endplate degeneration. Methods Pairs of axial biopsies (n = 58) spanning the entire vertebral body including both CEPs were collected from human cadaveric vertebrae with MC2. From one biopsy, the bone marrow directly adjacent to the CEP was analyzed with mass spectrometry. Differentially expressed proteins (DEPs) between MC2 and control were identified and bioinformatic enrichment analysis was performed. The other biopsy was processed for paraffin histology and BEP/CEP degenerations were scored. Endplate scores were correlated with DEPs. Results Endplates from MC2 were significantly more degenerated. Proteomic analysis revealed an activated complement system, increased expression of extracellular matrix proteins, angiogenic, and neurogenic factors in MC2 marrow. Endplate scores correlated with upregulated complement and neurogenic proteins. Discussion The inflammatory pathomechanisms in MC2 comprises activation of the complement system. Concurrent inflammation, fibrosis, angiogenesis, and neurogenesis indicate that MC2 is a chronic inflammation. Correlation of endplate damage with complement and neurogenic proteins suggest that complement system activation and neoinnervation may be linked to endplate damage. The endplate-near marrow is the pathomechanistic site, because MC2 occur at locations with more endplate degeneration. Conclusion MC2 are fibroinflammatory changes with complement system involvement which occur adjacent to damaged endplates

Impacts of priming on distinct immunosuppressive mechanisms of mesenchymal stromal cells under translationally relevant conditions

Abstract Background The multimodal properties of mesenchymal stromal cells (MSCs), particularly their ability to modulate immune responses is of high interest in translational research. Pro-inflammatory, hypoxic, and 3D culture priming are promising and often used strategies to improve the immunosuppressive potency of MSCs, but the underlying mechanisms are not well understood. Therefore, the aims of this study were (i) to compare the effects of pro-inflammatory, hypoxic, and 3D culture priming on the in vitro immunosuppressive potential of MSCs, (ii) to assess if immunosuppressive priming effects are temporally preserved under standard and translationally relevant culture conditions, and (iii) to investigate if the three priming strategies engage the same immunosuppressive mechanisms. Methods Functional in vitro T cell suppressive potency measurements were conducted to assess the impact of pro-inflammatory, hypoxic, and 3D culture priming on the immunosuppressive potential of human bone marrow-derived MSCs. Primed MSCs were either cultured under standard cell culture conditions or translationally relevant culture conditions, and their transcriptomic adaptations were monitored over time. Next-generation sequencing was performed to assess if different priming strategies activate distinct immunosuppressive mechanisms. Results (i) Pro-inflammatory, hypoxic, and 3D culture priming induced profound transcriptomic changes in MSCs resulting in a significantly enhanced T cell suppressive potential of pro-inflammatory and 3D culture primed MSCs. (ii) Priming effects rapidly faded under standard cell culture conditions but were partially preserved under translationally relevant conditions. Interestingly, continuous 3D culture priming of MSCs maintained the immunosuppressive potency of MSCs. (iii) Next-generation sequencing revealed that priming strategy-specific differentially expressed genes are involved in the T cell suppressive capacity of MSCs, indicating that different priming strategies engage distinct immunosuppressive mechanisms. Conclusion Priming can be a useful approach to improve the immunosuppressive potency of MSCs. However, future studies involving primed MSCs should carefully consider the significant impact of translationally relevant conditions on the preservation of priming effects. Continuous 3D culture could act as a functionalized formulation, supporting the administration of MSC spheroids for a sustainably improved immunosuppressive potency

Mutant SF3B1 promotes PDAC malignancy through TGF-β resistance

The splicing factor SF3B1 is recurrently mutated in various tumors, including pancreatic ductal adenocarcinoma (PDAC). The impact of the hotspot mutation SF3B1K700E on the PDAC pathogenesis, however, remains elusive. Here, we demonstrate that Sf3b1K700E alone is insufficient to induce malignant transformation of the murine pancreas, but increases aggressiveness of PDAC if it co-occurs together with mutated KRAS and p53. We further demonstrate that SF3B1K700E reduces epithelial–mesenchymal transition (EMT) and confers resistance to TGF-β1-induced cell death, and provide evidence that this phenotype is in part mediated through aberrant splicing of Map3k7. Taken together, our work suggests that SF3B1K700E acts as an oncogenic driver in PDAC through enhancing resistance to the tumor suppressive effects of TGF-β.Competing Interest StatementThe authors have declared no competing interest

Additional file 1 of Impacts of priming on distinct immunosuppressive mechanisms of mesenchymal stromal cells under translationally relevant conditions

Supplementary Material

Intervertebral disc microbiome in Modic changes: Lack of result replication underscores the need for a consensus in low-biomass microbiome analysis

Introduction: The emerging field of the disc microbiome challenges traditional views of disc sterility, which opens new avenues for novel clinical insights. However, the lack of methodological consensus in disc microbiome studies introduces discrepancies. The aims of this study were to (1) compare the disc microbiome of non-Modic (nonMC), Modic type 1 change (MC1), and MC2 discs to findings from prior disc microbiome studies, and (2) investigate if discrepancies to prior studies can be explained with bioinformatic variations. Methods: Sequencing of 16S rRNA in 70 discs (24 nonMC, 25 MC1, and 21 MC2) for microbiome profiling. The experimental setup included buffer contamination controls and was performed under aseptic conditions. Methodology and results were contrasted with previous disc microbiome studies. Critical bioinformatic steps that were different in our best-practice approach and previous disc microbiome studies (taxonomic lineage assignment, prevalence cut-off) were varied and their effect on results were compared. Results: There was limited overlap of results with a previous study on MC disc microbiome. No bacterial genera were shared using the same bioinformatic parameters. Taxonomic lineage assignment using "amplicon sequencing variants" was more sensitive and detected 48 genera compared to 22 with "operational taxonomic units" (previous study). Increasing filter cut-off from 4% to 50% (previous study) reduced genera from 48 to 4 genera. Despite these differences, both studies observed dysbiosis with an increased abundance of gram-negative bacteria in MC discs as well as a lower beta-diversity. Cutibacterium was persistently detected in all groups independent of the bioinformatic approach, emphasizing its prevalence. Conclusion: There is dysbiosis in MC discs. Bioinformatic parameters impact results yet cannot explain the different findings from this and a previous study. Therefore, discrepancies are likely caused by different sample preparations or true biologic differences. Harmonized protocols are required to advance understanding of the disc microbiome and its clinical implications.ISSN:2572-114