Altered early immune response after fracture and traumatic brain injury

Abstract

Introduction: Clinical and preclinical data suggest accelerated bone fracture healing in subjects with an additional traumatic brain injury (TBI). Mechanistically, altered metabolism and neuro-endocrine regulations have been shown to influence bone formation after combined fracture and TBI, thereby increasing the bone content in the fracture callus. However, the early inflammatory response towards fracture and TBI has not been investigated in detail so far. This is of great importance, since the early inflammatory phase of fracture healing is known to be essential for the initiation of downstream regenerative processes for adequate fracture repair.

Methods: Therefore, we analyzed systemic and local inflammatory mediators and immune cells in mice which were exposed to fracture only or fracture + TBI 6h and 24h after injury.

Results: We found a dysregulated systemic immune response and significantly fewer neutrophils and mast cells locally in the fracture hematoma. Further, local CXCL10 expression was significantly decreased in the animals with combined trauma, which correlated significantly with the reduced mast cell numbers.

Discussion: Since mast cells and mast cell-derived CXCL10 have been shown to increase osteoclastogenesis, the reduced mast cell numbers might contribute to higher bone content in the fracture callus of fracture + TBI mice due to decreased callus remodeling.

Keywords: fracture healing; inflammation; mast cells; polytrauma; traumatic brain injury.

Figure 1

Local gene expression of inflammatory mediators in the hematoma. RNase-free sections of the fractured bones were cut, RNA was isolated and gene expression analyzed by qPCR. Data are normalized to the housekeeping gene B2M and the Fx group, respectively. (A) Relative IL-6 gene expression at 6h and (B) 24h after injury. (C) Relative CXCL10 gene expression at 6h and (D) 24h after injury. (E) Relative IL-1beta gene expression at 6h and (F) 24h after injury. (G) Relative TNFalpha gene expression at 6h and (H) 24h after injury. (I) Relative IL-10 gene expression at 6h and (J) 24h after injury. (K) Experimental design. * indicates p-value below 0.05 for comparison between Fx and Fx+TBI.

Figure 2

Local protein expression of CXCL10 in the hematoma. Longitudinal sections of the fracture bones were cut and stained for CXCL10. Staining was quantified by positive pixel amount relative to the total pixel. (A) Local CXCL10 protein expression at 6h and (B) 24h after fracture. (C) Representative images from the fracture area at 6h after fracture. Scale bar = 50 µm. * indicates p-value below 0.05 for comparison between Fx and Fx+TBI.

Figure 3

Immune cell populations in the fracture hematoma. Longitudinal sections of the fracture bones were cut and stained for immune cell markers. (A) Ly6G⁺ neutrophil numbers at 6h and (B) 24h after injury. (C) F4/80⁺ macrophage numbers at 6h and (D) 24h after injury. (E) CD8⁺ T-lymphocyte numbers at 6h and (F) 24h after injury. (G) Mast cell numbers were determined in Toluidin blue staining at 6h and (H) 24h after injury. (I) Experimental design. * indicates p-value below 0.05 for comparison between Fx and Fx+TBI; ** indicates p-value below 0.01 for comparison between Fx and Fx+TBI.

Figure 4

Correlations between neutrophil/mast cell numbers and CXCL10 expression in the fracture hematoma during the early inflammatory phase. Correlation analysis by simple linear regression was performed between the parameters neutrophil numbers, mast cell numbers and local CXCL10 protein expression in all samples. (A) Neutrophil number/CXCL10 correlation. (B) Mast cell number/CXCL10 correlation. (C) Mast cell number/neutrophil number correlation. (D) Immunofluoresence double staining for mast cells (Avidin staining, red) and CXCL10 (green). DNA was counterstained with Hoechst (blue). Scale bar = 50 µm. E) Black box marked the area which is shown in (E). (F) Single fluorescent channels for Avidin (red) and CXCL10 (green). Scale bar = 50 µm.