69: Substance P-NK1R Activation in Tendon-Derived Cells as a Neuro-Immune Driver of Tendon Pain

Topic: Basic Science
Subtopic: Non-specific to any diagnosis (including studies in healthy animals), Developing methodology/technology, Neuroimmunology, Assessment and Measurement of Pain/Nociception

Background and Aims: Tendon pain is a hallmark feature of tendinopathy, yet mechanisms driving pain remain poorly defined[1]. Pain persists despite resolution of inflammation or structural restoration, suggesting that non-steroidal anti-inflammatory drugs and surgical repair may not address the etiology of pain. Substance P (SP) and neurokinin-1 receptor (NK1R) signaling contribute to inflammation and remodeling in multiple tissues[2]. In tendon-derived cells (TDCs), exogenous SP administration alters proliferation and promotes matrix-degrading remodeling. However, regulation of endogenous SP signaling in inflammatory conditions remains unclear[3,4,5]. The objective of this study is to assess SP-NK1R in TDCs and whether inflammatory macrophage cues increase SP and TNF-α.

Methods: 8-week-old female rat Achilles TDCs and bone marrow-derived macrophages were isolated. Macrophages were maintained as unpolarized controls (M0) or polarized to M1 using lipopolysaccharide and interferon gamma. Conditioned media (CM) was collected from M1 (M1-CM) and M0 (M0-CM). TDCs were treated with M1-CM, M0-CM, or media (non-treated controls) for up to 72 hours. Indirect cell viability was assessed by measuring metabolic activity (MTS assay). Secreted SP and pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α), were quantified using an enzyme-linked immunosorbent assay (ELISA). SP and NK1R expression were evaluated by immunofluorescence. Statistical significance was assessed using two-way ANOVA (p < 0.05).

Results: Metabolic activity peaked at 48 hours in non-treated TDCs and declined by 72 hours, while M1-CM treated TDCs showed a slower but sustained increase and remained highest at 72 hours. M1-CM significantly increased TNF-α secretion compared to unstimulated and M0-CM TDC controls. SP secretion was significantly elevated in M1-CM treated TDCs at 12, 48, and 72 hours and remained highest at 72 hours across all groups. Immunofluorescence confirmed baseline expression of SP and NK1R in non-treated TDCs, with SP localized to the cytoplasm and NK1R localized to the plasma membrane.

Conclusions: These findings demonstrate that TDCs actively respond to inflammatory macrophage signaling by increasing secretion of both TNF-α and the pain-related neuropeptide Substance P. Sustained SP production under M1 inflammatory conditions, combined with baseline NK1R expression and membrane localization, supports a mechanistic role for SP-NK1R signaling in tendon neuroimmune crosstalk. To our knowledge, this is the first study to demonstrate that inflammatory macrophage-derived cues directly increase SP secretion in TDCs and to validate SP/NK1R expression. This pathway may contribute to persistent tendon pain and maladaptive remodeling, representing a promising target for mechanism-based tendon pain therapies.

References: 1. Millar NL, et al. Tendinopathy. Nature Reviews Disease Primers. 2021.
2. Mashaghi A, et al. Neuropeptide substance P and the immune response. Cellular and Molecular Life Sciences. 2016.
3. Backman LJ, et al. Substance P Is a Mechanoresponsive, Autocrine Regulator of Human Tenocyte Proliferation. PLoS ONE. 2011.
4. Fong G, et al. Substance P Enhances Collagen Remodeling and MMP-3 Expression by Human Tenocytes. Journal of Orthopedic Research. 2013.
5. Wasker SVZ, et al. Is neurogenic inflammation involved in tendinopathy? A systematic review. BMJ Open Sport and Exercise Medicine. 2023.

Presenting Author: Dr. Brittany Taylor
Submitting Author: Mariah Turner

Senior Author: Dr. Brittany Taylor

IASP 2026 Global Year theme of Neuropathic Pain: Yes

• Folly Patterson: PhD - University of Florida
• Blanka Sharma: PhD - University of Florida
• Brittany Taylor: PhD - University of Florida