**Background:** Fracture healing involves a complex inflammatory phase, during which IL-17A plays a critical role. γδ T cells and Th17 cells are both sources of IL-17, but their origins and relative contributions to fracture repair were unknown. The gut microbiome, particularly segmented filamentous bacteria (SFB), is known to induce Th17 cell differentiation in the intestinal lamina propria. This study investigated whether gut-derived Th17 cells migrate to the fracture callus and contribute to healing, and whether this process depends on the microbiome and γδ T cell activation.
**Methods:** Closed femoral fractures were induced in 12-week-old female mice using a 3-point bending method with intramedullary pin stabilization. Multiple mouse strains were used: SFB+ and SFB- C57BL/6 mice (from Jackson Laboratory and Taconic), Tcrd-/- (γδ T cell-deficient), Tnf-/-, Il17a-EGFP reporter mice, and Kaede photoconvertible mice. Gut permeability was assessed by serum LPS and FITC-dextran absorption. Cell populations were analyzed by flow cytometry from callus tissue and Peyer's patches (PPs). T cell trafficking was tracked using Kaede mice (photoconversion of PPs) and adoptive transfer of EGFP+ Th17 cells. Interventions included: broad-spectrum antibiotics (ampicillin, vancomycin, neomycin, metronidazole), nonabsorbable antibiotics (neomycin, bacitracin), FTY720 (S1PR1 modulator, 5 μg/mL in drinking water), and neutralizing anti-CCL20 antibody (50 μg/mouse i.p. every other day). Fracture healing was assessed by micro-CT at days 14 and 21, and biomechanical torsion testing at day 35.
**Key Results:** In SFB+ mice, fractures increased callus Il17a, Tnf, Il1b, and Il6 transcripts at day 3 post-fracture (PF), which was blunted by antibiotics. In SFB- mice, only small increases in Tnf and Il1b were observed. Callus γδ T cells expanded by day 1 PF in both SFB+ and SFB- mice, independent of the microbiome. Th17 cells increased in PPs and callus only in SFB+ mice, peaking at day 3 PF, and this was blocked by antibiotics. In Tcrd-/- mice, fractures failed to expand Th17 cells, and gut permeability (measured by LPS and FITC-dextran) did not increase. Kaede mouse experiments showed that fractures increased homing of intestinal αβ T cells and Th17 cells to the callus, but not γδ T cells. Fractures increased CCL20 transcripts in callus cells (peak day 3), and this was dependent on TNF, as Tnf-/- mice showed no increase. Adoptive transfer of EGFP+ Th17 cells confirmed TNF-dependent homing to the callus. FTY720 prevented the fracture-induced increase in peripheral blood and callus Th17 cells and decreased callus Il17a transcripts, resulting in reduced BVc and BVc/TVc at day 14. Anti-CCL20 antibody prevented the increase in callus Th17 cells and decreased callus Il17a transcripts, also impairing healing at day 14. Micro-CT at day 14 showed that SFB+ mice had higher BVc (bone volume) and BVc/TVc (bone volume fraction) than SFB- mice, and Tcrd-/- mice had lower BVc and BVc/TVc than WT controls. At day 21, these differences resolved. However, biomechanical testing at day 35 showed higher torsion stiffness, yield torque, and ultimate torque in SFB+ vs SFB- mice.
**Clinical Implications:** This study reveals a gut-bone axis in fracture healing, where the gut microbiome (specifically SFB) drives Th17 cell expansion, and these cells migrate to the fracture site to improve healing. The findings suggest that broad-spectrum antibiotics, commonly used after trauma or orthopedic surgery, may impair fracture repair by depleting Th17-inducing bacteria. Conversely, Th17 cell-modulating bacteriotherapy could represent a novel strategy to enhance fracture healing. The use of FTY720 (an FDA-approved drug for multiple sclerosis) to block Th17 cell egress from the gut, and anti-CCL20 to block Th17 cell homing, both impaired healing, confirming the functional importance of this pathway. The study also highlights the critical role of γδ T cells in initiating the inflammatory cascade and increasing gut permeability, which then enables Th17 cell expansion.