Trigeminal neurons control immune-bone cell interactions and metabolism in apical periodontitis

Apical periodontitis (AP) is an inflammatory disease occurring following tooth infection with distinct osteolytic activity. Despite increasing evidence that sensory neurons participate in regulation of non-neuronal cells, their role in the development of AP is largely unknown. We hypothesized that Nav1.8+ nociceptors regulate bone metabolism changes in response to AP. Methods: A selective ablation of nociceptive neurons in Nav1.8Cre/ DTALox mouse line was used to evaluate the development and progression of AP using murine model of infection-induced AP. Micro-computed tomography examination was applied to quantify osteolytic lesions following induction of AP. Additionally, RT-PCR, RNAscope, and immunohistochemical (IHC) analysis were used to investigate the expression of immune cells, osteoblasts, and osteoclasts. Co-culture of trigeminal ganglia (TG) neurons from DTALox (control) and Nav1.8Cre/ DTALox mice with either IDG-SW3 or MC3T3-E1 osteoblast precursor cell lines or RAW264.7 murine macrophages were used to assess osteoblast and osteoclast function, by RNA sequencing, mineralization, and osteoclastic assays. Results: Ablation of Nav1.8+ nociceptors had earlier progression of AP with larger osteolytic lesions compared to the controls. IHC and RNAscope analysis demonstrated greater number of macrophages, T-cells, osteoclast and osteoblast precursors as well as an increased RANKL:OPG ratio at earlier time points among Nav1.8Cre/ DTALox mice. There was an increased expression of IL-1a and IL-6 within lesions of nociceptor-ablated mice. Further, co-culture experiments demonstrated that TG neurons promoted osteoblast mineralization and inhibited osteoclastic function. Conclusion: The findings suggest that TG Nav1.8+ neurons contribute to modulation of the AP development by delaying the influx of immune cells, promoting osteoblastic differentiation, and decreasing osteoclastic activities. This newly uncovered mechanism could become a therapeutic strategy for the treatment of AP and minimize the persistence of osteolytic lesions in refractory cases. Overall design: Trigeminal ganglia (TG) were harvested from Nav1.8Cre/ DTALox and Nav1.8Cre control mice, and cells were isolated via enzymatic digestion process, as previously described (Austah et al. 2019; Patwardhan et al. 2005). Single cell suspensions at the concentration of (~5x104 cells/insert) were seeded on 24-well plate polyethylene cell culture inserts (1µm-pore sizes) (Millipore-Sigma) precoated with 50µg/ml laminin (Sigma, MO) in growth medium containing Alpha Modified Eagle's Medium (aMEM) (Life Technologies, Carlsbad, CA), 10% heat-inactivated fetal bovine serum (HI-FBS) (Life Technologies, Carlsbad, CA), and 1X Glutamine-Penicillin- Streptomycin (Invitrogen, CA). TG cells were allowed to attach for 24 hours in 5% CO2 and 37ºC in the presence of 10µM cytosine ß-D- arabinofuranoside (Ara-C) (Sigma) to halt the non- neuronal cell proliferation, producing a neuron-enriched culture (Henriquez et al. 2013; Nicole et al. 2001). The control TG preparations are called TG-cre, and nociceptor-ablated cultures are called TG-DTA, where Nav1.8+, or sensory neuron specific (SNS), cells are ablated. The inserts containing either TG-cre or TG-DTA neuronal-enriched primary cultures were transferred to 24-well plates containing IDG-SW3 osteoblast precursor cells for osteoblast co-cultures.