IL-17RA Signaling in Prx1+ Mesenchymal Cells Influences Fracture Healing in Mice

Abstract

Fracture healing is a complex series of events that requires a local inflammatory reaction to initiate the reparative process. This inflammatory reaction is important for stimulating the migration and proliferation of mesenchymal progenitor cells from the periosteum and surrounding tissues to form the cartilaginous and bony calluses. The proinflammatory cytokine interleukin (IL)-17 family has gained attention for its potential regenerative effects; however, the requirement of IL-17 signaling within mesenchymal progenitor cells for normal secondary fracture healing remains unknown. The conditional knockout of IL-17 receptor a (Il17ra) in mesenchymal progenitor cells was achieved by crossing Il17ra^F/F mice with Prx1-cre mice to generate Prx1-cre; Il17ra^F/F mice. At 3 months of age, mice underwent experimental unilateral mid-diaphyseal femoral fractures and healing was assessed by micro-computed tomography (µCT) and histomorphometric analyses. The effects of IL-17RA signaling on the osteogenic differentiation of fracture-activated periosteal cells was investigated in vitro. Examination of the intact skeleton revealed that the conditional knockout of Il17ra decreased the femoral cortical porosity but did not affect any femoral trabecular microarchitectural indices. After unilateral femoral fractures, Il17ra conditional knockout impacted the cartilage and bone composition of the fracture callus that was most evident early in the healing process (day 7 and 14 post-fracture). Furthermore, the in vitro treatment of fracture-activated periosteal cells with IL-17A inhibited osteogenesis. This study suggests that IL-17RA signaling within Prx1+ mesenchymal progenitor cells can influence the early stages of endochondral ossification during fracture healing.

Keywords: IL-17A; cytokine; inflammation; periosteum; secondary bone healing.

Figure 1

Conditional deletion of Il17ra in Prx1+ cells leads to subtle changes in bone microarchitecture. (A) Prx1-cre; Il17ra^F/F (Il17ra cKO) were generated to conditionally delete Il17ra in Prx1+ mesenchymal cells by deleting exons 3 and 4 which leads to the early termination of translation. (B) Prx1-cre significantly decreased the Il17ra gene expression in isolated periosteal cells. (C) Immunohistochemistry shows less immunoreactivity of IL-17RA within day 14 fracture calluses and the periosteum of Il17ra cKO mice. Scale bar: 50 µm. Representative 3D images of (D) femur trabecular bone, (E) femur cortical bone, and (F) L3 vertebral body trabecular bone. µCT analysis showing (G) femur trabecular bone volume fraction (BV/TV), (H) femur trabecular thickness (Tb.Th), (I) femur cortical thickness (Ct.Th), (J) femur cortical porosity (Ct.Po), (K) L3 vertebral body trabecular bone volume fraction (BV/TV), and (L) L3 vertebral body trabecular thickness (Tb.Th). Abbreviations: PO, periosteum; Prom, promoter; cKO, conditional knockout. Data represent mean ± SD. Student’s unpaired t-test, * p < 0.05, ** p < 0.001.

Figure 2

Conditional deletion of Il17ra in Prx1+ mesenchymal progenitor cells delays healing. (A) Representative histological images and static histomorphometric analyses of fracture calluses at day 7, 14, and 18 post-fracture show changes in the composition of the callus across time. Scale bar: 100 µm. µCT volumetric analyses of fracture calluses at (B) day 14 and (C) day 18 post-fracture. Abbreviations: ns, not significant; BV/TV, bone volume fraction. Data represent mean ± SD. Student’s unpaired t-test, * p < 0.05.

Figure 3

Activation of IL-17RA signaling inhibits osteogenesis. (A) IL-17A at 20 and 50 ng/mL inhibited the gene expression of Runx2, Osx, Cola1, and Bglap in periosteal cells isolated from control mice. (B) IL-17A did not influence expression of Runx2, Osx, Cola1, and Bglap in Il17ra cKO periosteal cells. (C) Alizarin red-S staining and quantification shows less mineralization in IL-17A-treated control periosteal cells at days 14 and 21 of osteogenic differentiation, but there was no effect on mineralization by Il17ra cKO cells. Dashed line represented a fold-change of 1. Abbreviations: Diff, osteogenic differentiation; Osx, osterix; Cola1, collagen type 1; Bglap, osteocalcin. Data represent mean ± SD. The two-way ANOVA followed by Tukey’s multiple comparisons, * p < 0.05.

Figure 4

IL-17RA signaling promotes periosteal progenitor cell migration. Wound healing assay shows that IL-17A (20 ng/mL) promoted cell migration at 12 h. (Left): representative images of scratched areas marked by black lines. (Right): the semi-quantitative analysis of wound closure was determined by measuring the widths of the wounds. Data represent the mean ± SD. Two-way ANOVA followed by Tukey’s multiple comparisons. Values not sharing a common letter differ significantly, p < 0.05.