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. 2018 May;391(5):523-536.
doi: 10.1007/s00210-018-1483-7. Epub 2018 Mar 1.

Pharmacological Inhibition of IL-6 Trans-Signaling Improves Compromised Fracture Healing After Severe Trauma

Affiliations
Free PMC article

Pharmacological Inhibition of IL-6 Trans-Signaling Improves Compromised Fracture Healing After Severe Trauma

Kathrin Kaiser et al. Naunyn Schmiedebergs Arch Pharmacol. .
Free PMC article

Abstract

Patients with multiple injuries frequently suffer bone fractures and are at high risk to develop fracture healing complications. Because of its key role both in systemic posttraumatic inflammation and fracture healing, the pleiotropic cytokine interleukin-6 (IL-6) may be involved in the pathomechanisms of trauma-induced compromised fracture healing. IL-6 signals are transmitted by two different mechanisms: classic signaling via the membrane-bound receptor (mIL-6R) and trans-signaling via its soluble form (sIL-6R). Herein, we investigated whether IL-6 classic and trans-signaling play different roles in bone regeneration after severe injury. Twelve-week-old C57BL/6J mice underwent combined femur osteotomy and thoracic trauma. To study the function of IL-6, either an anti-IL-6 antibody, which inhibits both IL-6 classic and trans-signaling, or a soluble glycoprotein 130 fusion protein (sgp130Fc), which selectively blocks trans-signaling, were injected 30 min and 48 h after surgery. Bone healing was assessed using cytokine analyses, flow cytometry, histology, micro-computed tomography, and biomechanical testing. Selective inhibition of IL-6 trans-signaling significantly improved the fracture healing outcome after combined injury, as confirmed by accelerated cartilage-to-bone transformation, enhanced bony bridging of the fracture gap and improved mechanical callus properties. In contrast, global IL-6 inhibition did not affect compromised fracture healing. These data suggest that classic signaling may mediate beneficial effects on bone repair after severe injury. Selective inhibition of IL-6 trans-signaling might have therapeutic potential to treat fracture healing complications in patients with concomitant injuries.

Keywords: Bone fracture healing; Classic signaling; IL-6; Inflammation; Trans-signaling; Trauma.

Conflict of interest statement

Conflict of interest

Prof. Dr. Stefan Rose-John is an inventor on patents describing the function of sgp130Fc. He is also a shareholder of the CONARIS Research Institute (Kiel, Germany), which is commercially developing sgp130Fc proteins as therapeutics for inflammatory diseases. Georg H. Waetzig is an inventor on patents describing the function of sgp130Fc and an employee of CONARIS Research Institute AG. All other authors have no financial conflicts of interest.

Ethical approval

All applicable international, national, and institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Figures

Fig. 1
Fig. 1
Inflammatory mediators in the blood 3 h and 1 day after fracture (Fx) and combined fracture and thoracic trauma (Fx + TxT) in vehicle-, anti-IL-6 antibody-, and sgp130Fc-treated mice. Data are displayed as means ± standard deviation. a n = 5–10; b n = 5–6; c, d n = 6–10; e n = 6. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001. Data of untreated animals are presented in Supplemental Table 1
Fig. 2
Fig. 2
Hepatic acute-phase reaction 1 day after fracture (Fx) and combined fracture and thoracic trauma (Fx + TxT) in vehicle-, anti-IL-6- antibody-, and sgp130Fc-treated mice. Relative gene expression of a CRP C-reactive protein, b SAA serum amyloid A, and c CXCL1 chemokine (C-X-C motif) ligand 1 in the liver. Data are displayed as means ± standard deviation. n = 4–5. *p ≤ 0.05; **p ≤ 0.01
Fig. 3
Fig. 3
Pulmonary inflammation 3 h and 1 day after fracture (Fx) and combined fracture and thoracic trauma (Fx + TxT) in vehicle-, anti-IL-6 antibody-, and sgp130Fc-treated mice. a Representative images of hematoxylin and eosin (H&E) stained lungs of vehicle-treated mice after 3 h and b 1 day. c Representative images of lungs stained for neutrophils (Ly-6G+); arrowheads indicate positively stained cells. d Neutrophil (Ly-6G+) number in lung tissue. e IL-6 and f CXCL1 chemokine (C-X-C motif) ligand 1 concentrations in the broncho-alveolar lavage fluid after 3 h. Data are displayed as means ± standard deviation. D n = 5–6; E, F n = 8–9. *p ≤ 0.05; **p ≤ 0.01. Data of untreated animals are presented in Supplemental Table 1
Fig. 4
Fig. 4
Inflammatory mediators and immune cells in the fracture hematoma 3 h and 1 day after fracture (Fx) and combined fracture and thoracic trauma (Fx + TxT) in vehicle-, anti-IL-6 antibody-, and sgp130Fc-treated mice. Data are displayed as means ± standard deviation. a IL-6, b MCP-1 monocyte chemotactic protein 1, and c CXCL1 chemokine (C-X-C motif) ligand 1 concentrations after 3 h. d Proportion of neutrophils (CD11b+, Ly6G+), e macrophages (CD11b+, F4/80+), f B cells (CD19+), and g T cells (CD3+). ac n = 6–7, d n = 6–8, eg n = 7–8. **p ≤ 0.01, ***p ≤ 0.001
Fig. 5
Fig. 5
Histomorphometrical analyses of the fracture callus on day 10 after fracture (Fx) and combined fracture and thoracic trauma (Fx + TxT) in vehicle-, anti-IL-6 antibody-, and sgp130Fc-treated mice. a Representative histological images of the fracture callus stained with Safranin-O: Ct cortex, FG fracture gap. Boxed areas in a indicate the location of the higher magnified images in b. b Immunostaining of collagen type X. c Relative amount of bone and d cartilage in the fracture callus. e Proportion of collagen type X (ColX)-positive stained cartilage of the total cartilage determined by immunohistochemistry. Data are displayed as mean ± standard deviation. c, d n = 6; (e) n = 4–5. *p ≤ 0.05
Fig. 6
Fig. 6
Micro-computer tomography, histomorphometrical, and biomechanical analyses of the fracture callus on day 21 after fracture (Fx) and combined fracture and thoracic trauma (Fx + TxT) in vehicle-, anti-IL-6 antibody-, and sgp130Fc-treated mice. a Representative μCT three-dimensional reconstructions of the fracture callus. b Representative Giemsa-stained histological images of the fracture callus. c Bending stiffness of fractured femurs. Relative amount of d bone and e cartilage determined by histomorphometrical analyses. Data are displayed as mean ± standard deviation. n = 7–9 (c–e). **p ≤ 0.01
Fig. 7
Fig. 7
Scheme of IL-6 classic and trans-signaling and the proposed effects of fracture healing. In IL-6 classic signaling, IL-6 binds to its membrane-bound receptor (IL-6R), which then binds to a dimer of transmembrane glycoprotein 130 (gp130), inducing intracellular signal transduction. In IL-6 trans-signaling, IL-6 binds to its soluble receptor (sIL-6R), which is mainly shed by A Disintegrin and Metalloproteinase 17 (ADAM 17). The IL-6/sIL-6r complex then binds to the gp130 dimer. Our previous (Prystaz et al. 2017) and present results indicate that IL-6 classic signaling induces a balanced immune response and pro-regenerative effects on bone repair. In contrast, IL-6 trans-signaling, which is induced after severe injury, negatively affects fracture healing

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