TNFα Receptor 1 and Not Receptor 2 Affect Annulus Fibrosus and Nucleus Pulposus Response to Cytokine Challenge in a Rat Model

Abstract

Background: Painful intervertebral disc (IVD) degeneration (IVDD) involves chronic inflammation. Developing translational immunomodulatory strategies for IVDD is a priority with tumor necrosis factor alpha (TNFα) signaling an important target. TNFα binds to 2 receptors (TNFRs), with TNFR1 signaling promoting catabolism and apoptosis and TNFR2 signaling promoting anabolism and proliferation.

Methods: This study developed translational strategies to evaluate and modulate TNFR1 and TNFR2 signaling in rat in vivo and in vitro IVDD models. We used blocking antibodies, the TNFR2-activator Atsttrin, and small molecule inhibitors of TNFR1 to discern distinct TNFR1 and TNFR2-effects on annulus fibrosus (AF) and nucleus pulposus (NP) cells and to identify effective strategies for modulating specific TNFRs.

Results: TNFR1 was significantly increased with IVDD in vivo in the NP while TNFR2 was unaffected with very faint staining. TNFR1-specific small molecule inhibitors were effective in reducing catabolic effects of TNFα, highlighting the efficacy of this small molecule strategy for TNFR1 signaling modulation. Meanwhile, TNFR1 and TNFR2 inhibition in vitro was not effective with blocking antibodies on NP or AF cells, likely due to species-specificity of available blocking antibodies. Further, TNFR2 activation with Atsttrin was similarly ineffective, likely due to extremely low TNFR2 levels in both AF and NP cells.

Conclusions: TNFα receptor-specific signaling is important in rat IVDD in vivo and in vitro. TNFR1 inhibition was more effective with small molecules than using blocking antibodies. Low levels of TNFR2 in rat AF and NP cells and lack of efficacy of TNFR2-activator Atsttrin suggest native AF and NP cells have little capacity for TNFR2-dependent IVD repair.

Keywords: Atsttrin; Cell Culture; ErythrosineB; Physcion‐8‐O‐β‐D‐monoglucoside; inflammation; intervertebral disc degeneration; small molecules; tumor necrosis factor alpha.

FIGURE 1

TNFR1 is greater in AF than in NP and is affected by injury in NP. Sham (n = 4 AF, n = 4 NP) and injured (n = 6 AF, n = 6 NP) rat lumbar IVDs with TNFR1 (A, C) and TNFR2 (B, D) IHC with TolBlue counterstain. TNFR1 is greater in AF than in NP for Sham. TNFR1 is greater in Injury than Sham in NP. TNFR2 is lowly expressed and is not affected by IVD region or injury condition. (4‐6 biological donors; *p < 0.05).

FIGURE 2

TNFα challenge activates TNFR1 signaling with increased cleaved Caspase‐3 and modulates TNFR1 and TNFR2 expression. (A) TNFα receptors (TNFR1 and TNFR2) and their respective signaling pathways (created using the TNFα Pathway template on BioRender). Rat AF and NP cells in vitro subject to 24‐h TNFα challenge werer measured for, (B) Caspase‐3 (yellow) colocalized with DAPI (blue) to identify apoptotic cells (white arrows) quantified on ImageJ; (C) Caspase‐3 expression within the nucleus increased for AF but not for NP (n = 3–4). (D) qRT‐PCR (2^−ΔCT) of TNFα receptor expression (Tnfrsf1a: n = 6; Tnfrsf1b: n = 7) in AF and NP under basal conditions (normalized to Gapdh). (E) qRT‐PCR (2^−ΔΔCT) of TNFα receptor expression (Tnfrsf1a: n = 6; Tnfrsf1b: n = 7) in AF and NP under basal and TNFα conditions (normalized to Gapdh and basal). (B, C) (3 technical replicates; 3‐4 biological donors; *p < 0.05). (D, E) (3 technical replicates; 6‐7 biological donors; *p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 3

TNFα caused a pro‐inflammatory and catabolic shift in AF cells and NP cells with no effects of TNFR1 or TNFR2 antibody blocking. Gene expression of (A1, A2) pro‐inflammatory cytokines, (B1, B2) TNFα receptors, (C1, C2) catabolic, and (D1, D2) anabolic extracellular matrix markers in control and antibody conditions for AF (top) and NP (bottom) cells (3 technical replicates; 7 biological donors; normalized to Gapdh and basal; *p < 0.05, **p < 0.01).

FIGURE 4

TNFα caused a pro‐inflammatory and catabolic shift in AF and NP cells with no effect of Atsttrin, suggesting little/no effect of TNFR2 activation on NP or AF cells. Gene expression of (A1, A2) pro‐inflammatory cytokines, (B1, B2) TNFα receptors, (C1, C2) catabolic, and (D1, D2) anabolic extracellular matrix markers in control and Atsttrin treated conditions for AF (top) and NP (bottom) cells (3 technical replicates; 7 biological donors; normalized to Gapdh and basal; *p < 0.05, **p < 0.01).

FIGURE 5

Small molecule inhibition of TNFR1 partially rescued rat AF and NP cells from TNFα challenge. Gene expression of (A) pro‐inflammatory cytokines, (B) TNFα receptors, (C) catabolic, and (D) anabolic extracellular matrix markers in control and EryB or PMG treated conditions for AF cells (3 technical replicates; 4‐5 biological donors; normalized to Gapdh and basal; *: p < 0.05, **p < 0.01, ***p < 0.001).