Mechanically‐Induced Osteocyte/Th17 Cell Signaling and Subsequent Osteoclast Recruitment

Abstract

Mechanical loading of bone triggers bone modeling/formation, via osteocyte signaling. However, when mechanical loads of extreme force or repetition create tissue damage, osteocytes stimulate the targeted remodeling of the damaged bone matrix. We have previously reported an up regulation of the pro-inflammatory cytokine GRO/KC, T cell recruiting cytokines (IL-2, MCP-1), and an increase in mRANKL and osteoclast number within fatigue loaded rat ulna compared to non-loaded controls. These findings suggests a relationship between bone fatigue and the subsequent recruitment of both T cells and osteoclasts. We examined a novel, mechanically-induced signaling pathway between osteocytes and Th17 cells, and hypothesized that repetitive fluid shear stress (FSS) would induce osteocyte production of cytokines, IL-6 and TGF-β1, known to stimulate Th17 cell development and their secretion of osteoclastogenic cytokines, IL-17 and sRANKL. MLO-Y4 osteocyte-like cells were exposed to FSS (7±3 dynes and 10±3 dynes, at 1Hz) or Static control. The cells and medium were collected after 30 or 60 minutes of FSS exposure or static conditions. ELISA analysis of MLO-Y4 medium revealed greater IL-6 and TGF-β1 in FSS versus Static groups. MLO-Y4 medium also revealed less sRANKL in FSS versus Static groups. Murine spleen cells were then cultured in T cell supportive conditions and treated with 25% FSS or Static MLO-Y4 medium for 72 hours. ELISA analysis of T cell culture medium revealed greater IL-17 in T cell cultures treated with FSS medium versus Static medium. MLO-Y4 cells were then co-cultured with murine spleen cells in osteoclast supportive conditions and treated with 25% T cell medium (FSS TX or Static TX) for 10 days. ELISA and TRAP stain analysis of osteoclast cultures revealed greater sRANKL and TRAP+ osteoclasts in cultures treated with T cell medium (FSS TX) versus T cell medium (Static TX). To our knowledge these data are the first to support a novel, mechanically-induced osteocyte/Th17 cell signaling pathway. Our data illustrates that mechanically-stimulated MLO-Y4 cells secrete greater amounts of cytokines (IL-6 and TGF-β1) known to induce Th17 cell development, that T cell cultures treated with MLO-Y4 FSS medium produced greater amounts of IL-17, and that the addition of T cell medium (FSS TX) to osteoclast cultures leads to greater osteoclast formation; thus suggesting that repetitive loading of osteocytes potentiates Th17 cell activity and the subsequent recruitment of osteoclasts. Future in vivo studies examining the osteoclast response in relation to our osteocyte/Th17 cell signaling model would provide a new paradigm for targeted bone remodeling in response to mechanical stress. Support or Funding Information NASA Nebraska Space Grant A. Proposed mechanically-induced osteocyte/Th17 cell signaling model. B. Increased TRAP+ osteoclasts in cultures treated with T cell medium (FSS TX: 7+/−3 & 10+/−3) versus T cell medium (Static TX).