Ex vivo and In vitro proteomic approach to elucidate the relevance of IL‐4 and IL‐10 in Intervertebral disc pathophysiology

Background This study investigates the native presence and potential anabolic effects of interleukin (IL)-4 and IL-10 in the human intervertebral disc (IVD). Methods Human nucleus pulposus (NP) cells cultured in 3D from trauma and degenerate IVDs and NP explants were stimulated with 10 ng/mL IL-4, IL-10, or each in combination with 1 ng/mL IL-1β stimulation. The role of IL-4 and IL-10 in the IVD was evaluated using immunohistochemistry, gene expression, and Luminex multiplex immunoassay proteomics (73 secreted) and phosphoproteomics (21 phosphorylated proteins). Results IL-4, IL-4R, and IL-10R expression and localization in human cartilage endplate tissue were demonstrated for the first time. No significant gene expression changes were noted under IL-4 or IL-10 stimulation. However, IL-1β stimulation significantly increased MMP3, COX2, TIMP1, and TRPV4 expression in NP cells from trauma IVDs. Combined IL-4 and IL-1β treatment induced a significant increase in protein secretion of IL-1α, IL-7, IL-16, IL-17F, IL-18, IFNγ, TNF, ST2, PROK1, bFGF2, and stem cell factor exclusively in NP cells from degenerated IVDs. Conversely, the secretome profile of explants revealed an IL-4–mediated decrease in CXCL13 following treatment with IL-1β. Combined IL-10 and IL-1β treatment increased neurotrophic growth factor secretion compared with IL-10 baseline. Conclusions The NP cell phenotype affects the pleiotropic role of IL-4, which can induce a pro-inflammatory response in the presence of catabolic stimuli and enhance the effects of IL-1β in degenerated IVDs. Environmental factors, including 3D culture and hypoxia, may alter IL-4's role. Finally, IL-10's potential neurotrophic effects under catabolic stimuli warrant further investigation to clarify its role in IVD degeneration.