Op0083 infectious and Autoinflammatory Modic Type 1 Changes Have Different Pathomechanisms

Abstract

Background:Modic type 1 changes (MC1) are vertebral bone marrow (BM) edema that associate with non-specific low back pain (LBP). Two etiologies have been described. In the infectious etiology the anaerobic aerotolerant Cutibacterium acnes (C. acnes) invades damaged intervertebral discs (IVDs) resulting in disc infection and endplate damage, which leads to the evocation of an immune response. In the autoinflammatory etiology disc and endplate damage lead to the exposure of immune privileged disc cells and matrix to leukocytes, thereby evoking an immune response in the BM. Different etiologies require different treatment strategies. However, it is unknown if etiology-specific pathological mechanisms exist.Objectives:The aim of this study was to identify etiology-specific dysregulated pathways of MC1 and to perform in-depth analysis of immune cell populations of the autoinflammatory etiology.Methods:BM aspirates and biopsies were obtained from LBP patients with MC1 undergoing spinal fusion. Aspirates/biopsies were taken prior screw insertion through the pedicle screw trajectory. From each patient, a MC1 and an intra-patient control aspiration/biopsy from the adjacent vertebral level was taken. If C. acnes in IVDs adjacent to MC1 were detected by anaerobic bacterial culture, patients were assigned to the infectious, otherwise to the autoinflammatory etiology.Total RNA was isolated from aspirates and sequenced (Novaseq) (infectious n=3 + 3, autoinflammatory n=5 + 5). Genes were considered as differentially expressed (DEG) if p-value < 0.01 and log2fc > ± 0.5. Gene ontology (GO) enrichment was performed in R (GOseq), gene set enrichment analysis (GSEA) with GSEA software.Changes in cell populations of the autoinflammatory etiology were analyzed with single cell RNA sequencing (scRNAseq): Control and MC1 biopsies (n=1 + 1) were digested, CD45+CD66b- mononuclear cells isolated with fluorescence activated cell sorting (FACS), and 10000 cells were sequenced (10x Genomics). Seurat R toolkit was used for quality-control, clustering, and differential expression analysis.Transcriptomic changes (n=5 + 5) of CD45+CD66b+ neutrophils isolated with flow cytometry from aspirates were analyzed as for total bulk RNAseq. Neutrophil activation (n=3 + 3) was measured as CD66b+ expression with flow cytometry. CD66bhigh and CD66blow fractions in MC1 and control neutrophils were compared with paired t-test.Results:Comparing MC1 to control in total bulk RNAseq, 204 DEG in the autoinflammatory and 444 DEG in the infectious etiology were identified with only 67 shared genes (Fig. 1a). GO enrichment revealed “T-cell activation” (p = 2.50E-03) in the autoinflammatory and “complement activation, classical pathway” (p=1.1E-25) in the infectious etiology as top enriched upregulated biological processes (BP) (Fig 1b). ScRNAseq of autoinflammatory MC1 showed an overrepresentation of T-cells (p= 1.00E-34, OR=1.54) and myelocytes (neutrophil progenitor cells) (p=4.00E-05, OR=2.27) indicating an increased demand of these cells (Fig. 1c). Bulk RNAseq analysis of neutrophils from the autoinflammatory etiology revealed an activated, pro-inflammatory phenotype (Fig 1d), which was confirmed with more CD66bhigh neutrophils in MC1 (+11.13 ± 2.71%, p=0.02) (Fig. 1e).Figure 1.(a) Venn diagram of DEG from total bulk RNAseq (b) Top enriched upregulated BP of autoinflammatory (left) and infectious (right) etiology (c) Cell clustering of autoinflammatory MC1 BM (d) Enrichment of “inflammatory response” gene set in autoinflammatory MC1 neutrophils (e) Representative histogram of CD66b+ expression in MC1 and control neutrophils.Conclusion:Autoinflammatory and infectious etiologies of MC1 have different pathological mechanisms. T-cell and neutrophil activation seem to be important in the autoinflammatory etiology. This has clinical implication as it could be explored for diagnostic approaches to distinguish the two MC1 etiologies and supports developing targeted treatments for both etiologies.Disclosure of Interests:None declared

Role of C‐reactive protein in the bone marrow of Modic type 1 changes

“…Recent in-depth analysis of MC1 bone marrow provide now new evidence that the hematopoietic elements in MC1 are indeed in an inflammatory state. Activation and accumulation of neutrophils, and T-cells have been reported 39 . If these mechanisms include the release of pro-inflammatory cytokines and if they relate to CRP and necrosis remains unknown.…”

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

“…In MC1, neutrophilic infiltrates were found, 15 maturation of neutrophils is dysregulated, 15 and neutrophils are activated. 36 In contrast, in MC2, no neutrophilic infiltrates were found and the neutrophil progenitor pool is depleted. 12 , 15 Hence, in the transition from MC1 to MC2, neutrophils must be cleared.…”

“… 32 , 33 In MC, occult infection of the disc as well as autoimmune reactions of the bone marrow against disc material are potential etiologies. 2 , 17 , 34 , 35 , 36 Hence, involvement of MAC in MC pathophysiology is plausible.…”

“…MAC can either be embedded in membranes resulting in membrane lysis of pathogens, or it activates leukocytes as a consequence of autoantibody‐antigen complex formation in autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus 32,33 . In MC, occult infection of the disc as well as autoimmune reactions of the bone marrow against disc material are potential etiologies 2,17,34–36 . Hence, involvement of MAC in MC pathophysiology is plausible.…”

Should Degenerated Intervertebral Discs of Patients with Modic Type 1 Changes Be Treated with Mesenchymal Stem Cells?

“…Moreover, suppression of discal inflammation might terminate nerve ingrowth into the IVD, as IL-1β was shown to stimulate the expression of vascular endothelial growth factor (VEGF), NGF, and brain-derived neurotrophic factor (BDNF) in degenerative IVDCs, resulting in angiogenesis and innervation [ 148 ]. Microvascular blood vessels expressing NGF were shown to enter painful IVDs from the adjacent bone marrow through the CEP [ 149 ]. These microvascular blood vessels were accompanied by neurotrophic receptor tyrosine kinase 1 (TrkA)-expressing nerve fibers.…”