Macrophages Down-Regulate Gene Expression of Intervertebral Disc Degenerative Markers Under a Pro-inflammatory Microenvironment

Abstract

Low back pain is a highly prevalent clinical problem and intervertebral disc (IVD) degeneration is now accepted as the major pathophysiological mechanism responsible for this condition. Accumulating evidence suggests that inflammation plays a crucial role in the progression of human IVD degeneration, with macrophages being pointed as the key immune cell players in this process since their infiltration in degenerated IVD samples has been extensively demonstrated. Since they are highly plastic, macrophages can play different roles depending on the microenvironmental cues. The study of inflammation associated with IVD degeneration has been somehow neglected and one of the reasons is related with lack of adequate models. To overcome this, we established and characterized a new model of IVD organ culture under pro-inflammatory conditions to further dissect the role of macrophages in IVD associated immune response. For that, human monocyte-derived macrophages were co-cultured either with bovine caudal IVD punches in the presence of the pro-inflammatory cytokine IL-1β, or IVD-conditioned medium (CM), to investigate how IVD-produced factors influence macrophage phenotype. After 72 h, metabolic activity, gene expression and cytokine profile of macrophages and IVD cells were measured. Our results show that macrophages and IVDs remain metabolically active in the presence of IL-1β, significantly upregulate CCR7 gene expression and increase production of IL-6 on macrophages. When treating macrophages with IL-1β-IVD-CM, CCR7 upregulation follows the same trend, while for IL-6 an opposite effect was observed. On the other hand, macrophages interfere with IVD ECM remodeling, decreasing MMP3 expression and downregulating aggrecan and collagen II gene expression in the presence of IL-1β. Overall, the co-culture model established in this study can be considered a suitable approach to address the cellular and molecular pathways that regulate macrophage-IVD crosstalk, suggesting that degenerated IVD tissue tends to polarize human macrophages toward a more pro-inflammatory profile, which seems to aggravate IVD degeneration. This model could be used to improve the knowledge of the mechanisms that link IVD degeneration and the immune response.

The Immune Privilege of the Intervertebral Disc: Implications for Intervertebral Disc Degeneration Treatment

“…By co-culture of autologous or allogeneic peripheral blood mononuclear cells and NP cells, Stich et al found elevated immune cell proliferation levels in 3Dcultures than 2D-cultures, and a general trend to higher responses for NP cells from severely degenerated IVD tissue [47], indicating that autoimmune response could vary depending on different cell cultures and degeneration degrees. More recently, Silva et al established a model of IVD organ culture, found that human macrophages could be polarized toward a more pro-inflammatory profile by degenerated IVD tissue, and interfere with IVD ECM remodeling by downregulating aggrecan and collagen II gene expression in the presence of IL-1β [48]. These findings showed that with the breakdown of the BNB, the exposed NP tissue could induce autoimmune response, which stimulates both immunocytes activation and inflammatory factors infiltration ( Figure 2B).…”

“…Also, in a co-cultures system, Yang et al showed that AF or NP cells exposed to macrophages upregulated the expression of pro-inflammatory mediators [50]. As for the ECM, studies have found that immunocytes could interfere with IVD ECM remodeling and downregulate aggrecan and collagen II expression [48]. In addition, the expression of matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) were increased in IVD with autoimmune reaction, causing the degradation of ECM [51,52].…”

Articular Repair/Regeneration in Healthy and Inflammatory Conditions: From Advanced In Vitro to In Vivo Models

“…IVDs culture under proinflammatory conditions (puncture + IL-1β) with macrophages Macrophages interfere in the IVD ECM remodeling and exhibit a more-proinflammatory profile in contact with IVD degenerated tissue. [143] vivo processes, [116b] low oxygenation and nutrition, [118] or proinflammatory cues. [116c] Ex vivo studies use a variety of tissue sources, including human, bovine, sheep, rabbit, rat, and mouse, among others and are commonly safe and easy to manipulate.…”

An Injectable Hyaluronan–Methylcellulose (HAMC) Hydrogel Combined with Wharton’s Jelly-Derived Mesenchymal Stromal Cells (WJ-MSCs) Promotes Degenerative Disc Repair

“…As the stage of degeneration progresses, the production of pro-inflammatory molecules, including tumor necrosis factor (TNF)-α, interferon gamma (IFN-γ), and interleukin (IL-1β, IL-6, IL-1α and IL-2) increases [ 5 ]. These molecules can be produced by both IVD cells and immune cells, such as macrophages [ 6 ], and are known to be associated with discogenic back pain [ 6 ]. IVD degeneration is characterized by the loss of IVD cells and extracellular matrix (ECM) such as aggrecan and collagen type II, with the upregulation of matrix metalloproteinases (MMPs) and inflammatory mediators leading to progressive and irreversible damage of IVD structure [ 1 , 3 , 4 , 6 , 7 , 8 ].…”

“…These molecules can be produced by both IVD cells and immune cells, such as macrophages [ 6 ], and are known to be associated with discogenic back pain [ 6 ]. IVD degeneration is characterized by the loss of IVD cells and extracellular matrix (ECM) such as aggrecan and collagen type II, with the upregulation of matrix metalloproteinases (MMPs) and inflammatory mediators leading to progressive and irreversible damage of IVD structure [ 1 , 3 , 4 , 6 , 7 , 8 ]. Eventually, IVD degeneration can lead to the onset of additional spinal conditions, including disc herniation, spinal stenosis, facet joint osteoarthritis, and spondylolisthesis [ 1 , 3 , 9 , 10 ].…”