gms | German Medical Science

Objectives: Neutrophils are within the first cells that are recruited to the fracture site. They phagocyte pathogens, produce extracellular traps, reactive oxygen species, tissue proteases and many inflammatory mediators and are important for the healing process. Notably, they are a main source of interleukin-6 (IL-6) and the soluble IL-6 receptor (sIL-6R), which can induce IL-6 classic and trans-signaling. Previously, we found that IL-6 classic signaling regulates neutrophil infiltration to the fracture hematoma and is essential for bone repair, whereas IL6 trans-signaling contributes to compromised healing after severe trauma. However, the underlying mechanisms remain unclear. Here, we studied if neutrophils regulate the immune response after fracture and the healing process via the modulation of IL-6 signaling.

Methods: To that end, we generated mice with a neutrophil (B6.Ly6G(tm2621(Cre-tdTomato)Arte) specific deletion of IL-6 (B6.Il6^tm1.1Jho) or IL-6R (B6.SJL-Il6ra^tm1.1Drew), respectively. Firstly, we analyzed the bone phenotype of both mouse lines (males, 12- and 36-weeks old) by biomechanical testing, µCT analysis, and histomorphometry (n=5–7/group). To investigate their immune phenotype, we performed flow cytometry of bone marrow (BM) and spleen. To study fracture healing, male mice (12-weeks old) received a standardized femur osteotomy stabilized by an external fixator. The inflammatory response was analyzed after 6h by cytokine multiplex, and bone formation after 21d by biomechanical testing and µCT analysis (n=5–8/group). Cre- littermates served as controls. Statistics: ANOVA/post hoc Fishers LSD, p<0.05.

Results and conclusion: Neutrophil specific deletion of IL-6 or IL-6R did not influence the physiological bone turnover of the mice at neither age, as the biomechanical properties and the structural and cellular bone parameters in the cortical and trabecular bone did not differ compared to controls. However, neutrophil specific deletion of IL-6 significantly reduced macrophages in the BM (3.9 vs 2.8% living cells, p=0.013), and neutrophil specific deletion of IL-6R significantly diminished neutrophils (BM: 52 vs 38% living cells) and macrophages, but increased T lymphocytes (spleen: 32 vs 37% living cells) both in the BM and spleen compared to controls. The immune response towards fracture was not affected by the deletion of neither IL-6 or IL-6R in neutrophils, as cytokine levels in the fracture hematoma or serum did not significantly differ 6h after fracture. Also, the biomechanical properties of the fractured femurs and the structural bone parameters on d21 were not different compared to controls.

Our results imply that neutrophil-induced IL-6 signaling does not play a critical role in physiological bone turnover or fracture healing. However, it might be important for the regulation of the immune status. This could be critical for fracture healing under conditions of aberrant inflammation, e.g., after polytrauma or in osteoporotic bone, and needs further investigation.