Macrophage Subtypes Distinctly Impact Intervertebral Disc Inflammation and Extracellular Matrix Composition

Intervertebral disc (IVD) degeneration is characterized by changes in extracellular matrix (ECM) composition and an inflammatory microenvironment, including macrophage recruitment. While inflammation is known to amplify ECM degradation, the individual roles of IVD cells and macrophages, and contributions of crosstalk are unknown. Additionally, the impacts of polarized pro-inflammatory M1 or anti-inflammatory M2 macrophages on the IVD are unknown, highlighting a need for an in vitro macrophage-IVD explant co-culture system.

IVDs were dissected from the lumbar spine of rats and cultured encapsulated in agarose. Primary bone marrow-derived macrophages were isolated and polarized to M1 or M2. IVD explants were cultured for 14 days with or without TNFα. M1 or M2 macrophages were co-cultured with unstimulated or TNFα-stimulated IVDs. Changes in ECM were evaluated using glycosaminoglycan (GAG) loss into media, IVD GAG and collagen content, and histology. Inflammatory responses were assessed by measuring nitric oxide (NO) and cytokines (TNFα, IL-6, IL-13, CCL2) in media supernatants.

TNFα stimulation of IVDs increased NO, TNFα, and CCL2, indicating an impact on inflammation. TNFα stimulation of IVDs also affected IVD ECM, with changes observed in both GAG and collagen. M1 and M2 co-culture counteracted TNFα-induced changes in collagen content, while macrophage subtypes distinctly modulated inflammation. Specifically, M1 macrophages increased inflammatory cytokines (NO, IL-6), while M2 macrophages decreased pro-inflammatory (NO, TNFα) and increased anti-inflammatory (IL-13) cytokines. Macrophage-IVD crosstalk had unique effects on IL-6 and IL-13 levels compared to IVDs or macrophages cultured alone.

Inflammatory stimulation of IVDs and co-culture with macrophages in vitro impacted both inflammation and ECM composition. Both macrophage subtypes reversed TNFα-induced changes in collagen content, while M1 and M2 macrophages had detrimental and protective effects on inflammation, respectively. The macrophage-IVD explant co-culture model in this study provides a novel system for evaluating crosstalk exclusively through paracrine signaling.