TNFR1-mediated senescence and lack of TNFR2-signaling limit human intervertebral disc cell repair potential in degenerative conditions

Abstract

Objective: To identify mechanisms and treatment targets in painful intervertebral disc (IVD) degeneration (IVDD) progression with a focus on pro-inflammatory tumor necrosis factor-alpha (TNFα)-receptor-1 (TNFR1) and pro-reparative TNFα receptor-2 (TNFR2) signaling.

Design: IVDD tissues and cells from IVDD and autopsy subjects were analyzed with single-cell RNA-sequencing to identify cell populations expressing TNFR1 and TNFR2, and multiplexed array to identify inflammatory proteins in IVDD conditioned media (CM). Bulk RNA-seq evaluated inflammatory and cell cycle states of human annulus fibrosus (hAF) cells challenged with CM. hAF cell responses to TNFR1 and TNFR2 modulation were evaluated by treatment with TNFR1- and TNFR2-blocking antibodies and TNFR2-activator Atsttrin.

Results: IVDD CM chemokines and cytokines were expressed primarily by a small macrophage population and at low levels by native IVD cells. CM-treated hAF cells exhibited TNFα-signaling responses with reduced metabolic rates (MTT: 0.75 [95%CI:0.67 to 0.82]), limited inflammatory responses (inferred from heatmap of 50 differentially expressed genes), and senescence (10.4% SA-β-Gal+ cells [95%CI:6.99 to 13.8]). TNFR1-inhibition sufficiently restored hAF cell metabolism to enable robust pro-inflammatory responses to the complex IVDD CM cytokine mixture (multiple assays,). TNFR2-staining was limited on human IVD cell membranes and TNFR2 modulation had no effect on hAF cells, together suggesting a lack of TNFR2-signaling in native IVD cells.

Conclusions: Secreted proteins from IVDD CM caused hAF cells to have reduced metabolic rates, attenuated inflammatory responses, and senescence indicating a TNFR1-dominated response with metabolic impairment. Meanwhile, human IVD cells lacked reparative TNFR2-signaling since its modulation caused no effects, to suggest enhanced TNFR2-signaling in IVD repair may need recruitment or delivery of macrophages or other TNFR2-expressing cells.

Keywords: Atsttrin; Intervertebral disc degeneration; Senescence; Single-cell RNA-sequencing; TNFR1; TNFR2.