(L-446) Axial Compression of Tibia Increases Marrow Macrophage and T-Cell Subsets in a Load-Dependent Manner, but Does not Differentially Regulate Muscle Gene Expression.

  Immune system dysregulation is linked to negative changes in skeletal muscle and bone mass and strength. 1 Autoinflammatory diseases such as rheumatoid arthritis is known to cause the degeneration of skeletal muscle. Axial bone loading is a known anabolic treatment to increase bone mass via WNT-β-catenin upregulation.2, 3 Although, WNT-β-catenin upregulation is known to altering immune cell populations within the bone marrow4 and inducing skeletal muscle hypertrophy8, the role of tibial loading in inducing these same alterations is unknown. Bone-muscle crosstalk, which describes the mutual regulation of bone and muscle tissue through mechanical and biochemical signals, is similarly underexplored as a potential regulator of the immune system. Skeletal muscle is known to modulate immune cell response through the release of anti-inflammatory cytokines such as IL-6 during exercise7, however, whether axial compression of the bone will induce any differential changes in skeletal muscle gene expression is unknown8.  

Male 16-week C57BL/6J mice underwent in vivo tibial loading (right limb only) under anesthesia for three consecutive days (60 cycles, 2Hz triangle waveform) from 0.5N to 4N or 9N similar to waveforms used previously.5, 6 Mice undergoing anesthesia and preload (-0.5N) but no dynamic loading served as sham controls. All mice received buprenorphine ER lab (0.5mg/kg, s.c.) throughout loading. Mice were euthanized one day after cessation of loading and tibial cortical bone and the gastrocnemius underwent qPCR while marrow underwent flow cytometry analysis. Data analysis used 2-WAY Repeated measures ANOVA for effect of load (right versus left limb) and load level (sham, 4N, 9N).

Results: 9N loaded limbs showed significantly increased Col1a1 expression relative to nonloaded control limbs (Figure 1A). In addition, the 9N loaded group showed decreased SOST expression in both tibias compared to other load levels demonstrating increased anabolism. Macrophages (F4/80+/CD11c- of CD45+) and T-Cells (CD3+ of CD45+) showed significant dose-dependent increases in marrow populations based on load magnitude and limb (p< 0.05; Figure 2). Mast cells were elevated in loaded limbs overall they showed no significant changes due to load level and B-cells were unaffected by any parameters (p >0.05). Load did not significantly change the expression of key regulatory muscle genes compared to non loaded control limbs, however, Igf1 expression may be load responsive systemically. (p >0.05; Figure 1B).

Conclusions: Our qPCR results suggest that tibial loading, although anabolic to bone, may not significantly affect muscle differentiation or degradation. However, tibial loading induces significant upregulation of macrophages and T-cells in bone marrow in a dose-dependent manner.Previous results from our group have shown that these same cell immune cell populations are most differentially responsive to orthopedic implant substrate.4 These results suggest that macrophages and T-cells are highly mechanoresponsive. 

Discussion: The mechanoresponsiveness of macrophages and T-cells requires future research to determine their dispensability in the anabolic responses to bone loading. Future research will examine the expression of immunoregulatory cytokines such as IL-6 within the bone and gastrocnemius to different load levels to elucidate how mechanical loading may alter immune system dynamics.

Tsukasaki, M. and H. Takayanagi, Osteoimmunology: evolving concepts in bone–immune interactions in health and disease. Nature Reviews Immunology, 2019. 19(10): p. 626-642.

2. Chermside-Scabbo, C.J., et al., Old Mice Have Less Transcriptional Activation But Similar Periosteal Cell Proliferation Compared to Young-Adult Mice in Response to in vivo Mechanical Loading. J Bone Miner Res, 2020. 35(9): p. 1751-1764.

3. Lawson, L.Y., et al., Osteoblast-Specific Wnt Secretion Is Required for Skeletal Homeostasis and Loading-Induced Bone Formation in Adult Mice. J Bone Miner Res, 2022. 37(1): p. 108-120.

4. Avery, D., et al., Canonical Wnt signaling enhances pro-inflammatory response to titanium by macrophages. Biomaterials, 2022. 289: p. 121797.

5. Sun, D., et al., Evaluation of loading parameters for murine axial tibial loading: Stimulating cortical bone formation while reducing loading duration. J Orthop Res, 2018. 36(2): p. 682-691.

6. Main, R.P., et al., Murine Axial Compression Tibial Loading Model to Study Bone Mechanobiology: Implementing the Model and Reporting Results. Journal of Orthopaedic Research, 2020. 38(2): p. 233-252.

7.      Rogeri, P.S., et al., Crosstalk Between Skeletal Muscle and Immune System: Which roles do IL-6 and Glutamine Play? Front Physiol, 2020. 11: pg. 582258.

8.           Maltzahn, J.V., et al., Wnt Signalling in Myogenesis. Trends Cell Biol, 2012. 22(11): p.602-609.

9.           Londhe, P. and D. C. Guttridge, Inflammation induced loss of skeletal muscle. Bone, 2015. 80: pg. 131-142.