# Non-neuronal cholinergic stimulation favors bone mass accrual

## TL;DR

Stimulating non-neuronal cholinergic receptors in immune cells increases bone mass by boosting osteoblast activity and IL-17 levels.

## Contribution

This study reveals a novel peripheral neuro-immune pathway linking cholinergic stimulation to bone formation via IL-17.

## Key findings

- Neostigmine treatment increased bone mass in mice by enhancing osteoblast proliferation and bone formation.
- Neostigmine accelerated bone defect healing in rats and increased serum IL-17 levels.
- IL-17, but not acetylcholine or neostigmine, directly promoted osteoblast proliferation in vitro.

## Abstract

Non-neuronal cholinergic receptors are expressed in immune cells and their stimulation has been shown to regulate the secretion of several cytokines. Some of these cytokines, such as interleukin-17 (IL-17), IL-23, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), are known to regulate bone mass. Accordingly, we hypothesize that stimulating cholinergic receptors in non-neuronal cells, such as immune cells, promotes bone mass accrual.

To test this hypothesis, we used neostigmine, a drug that increases acetylcholine levels by inhibiting acetylcholinesterase activity in peripheral tissues. Female C57BL/6 mice were treated with neostigmine for six weeks, and μCT, histomorphometry, Raman spectroscopy, X-ray diffraction, and mechanical testing were used to analyze bone parameters. A rat model was used to assess bone defect healing and implant osseointegration. Serum cytokines were measured by ELISA, and IL-17 effects on osteoblast proliferation were evaluated in vitro.

Here, we show that 6 weeks of neostigmine treatment promotes bone mass accrual in endochondral bones of both the axial and appendicular skeleton in mice. Moreover, the administration of neostigmine for 2 weeks accelerated the healing process of the surgically induced bone defects in rats. The body mass index, body weight, visceral fat pad weight and epinephrine levels in the neostigmine-treated mice were similar to those of saline-treated mice, indicating that neostigmine favored bone mass accrual by acting peripherally rather than centrally. The increased bone mass in the neostigmine-treated mice was caused by an increase in osteoblast proliferation and bone formation rate. We also observed an increase in circulating immunocytokine IL-17 levels in the neostigmine-treated mice. Statistical analysis showed that the increase in serum IL-17 level was associated with the increase in osteoblast number. In agreement with our findings from the in vivo experiments, IL-17 treatment increased the proliferation of MC3T3.E1 preosteoblasts in vitro, while acetylcholine or neostigmine did not have any significant effect.

Taken together, our findings indicate that peripheral cholinergic stimulation promotes bone mass accrual, in part through IL-17–mediated osteoblast activity. Although the evidence is correlative, these results highlight a potential neuro-immune pathway and suggest new therapeutic directions for enhancing bone formation and regeneration.