Hindlimb unloading causes regional loading-dependent changes in osteocyte inflammatory cytokines that are modulated by exogenous irisin treatment

Abstract

Disuse-induced bone loss is characterized by alterations in bone turnover. Accruing evidence suggests that osteocytes respond to inflammation and express and/or release pro-inflammatory cytokines; however, it remains largely unknown whether osteocyte inflammatory proteins are influenced by disuse. The goals of this project were (1) to assess osteocyte pro-inflammatory cytokines in the unloaded hindlimb and loaded forelimb of hindlimb unloaded rats, (2) to examine the impact of exogenous irisin during hindlimb unloading (HU). Male Sprague Dawley rats (8 weeks old, n = 6/group) were divided into ambulatory control, HU, and HU with irisin (HU + Ir, 3×/week). Lower cancellous bone volume, higher osteoclast surfaces (OcS), and lower bone formation rate (BFR) were present at the hindlimb and 4th lumbar vertebrae in the HU group while the proximal humerus of HU rats exhibited no differences in bone volume, but higher BFR and lower OcS vs. Con. Osteocyte tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), RANKL, and sclerostin were elevated in the cancellous bone of the distal femur of HU rats vs. Con, but lower at the proximal humerus in HU rats vs. Con. Exogenous irisin treatment increased BFR, and lowered OcS and osteocyte TNF-α, IL-17, RANKL, and sclerostin in the unloaded hindlimb of HU + Ir rats while having minimal changes in the humerus. In conclusion, there are site-specific and loading-specific alterations in osteocyte pro-inflammatory cytokines and bone turnover with the HU model of disuse bone loss, indicating a potential mechanosensory impact of osteocyte TNF-α and IL-17. Additionally, exogenous irisin significantly reduced the pro-inflammatory status of the unloaded hindlimb.

Keywords: Cytokines; Physiology.

Fig. 1. Cancellous static histomorphometry of the proximal tibia metaphysis (PT), femoral neck (FN), fourth lumbar vertebrae (L4), and proximal humerus (PH).

a Cancellous bone volume was lower in both HU groups compared to Con at the proximal tibia, femoral neck, and L4. There were not differences in cancellous bone volume at the proximal humerus. b Osteoclast-covered surfaces were higher in the HU group at the proximal tibia, femoral neck, and L4 compared to Con and HU + Ir. At the proximal humerus osteoclast-covered surfaces were higher in Con compared to both HU groups. c Osteoid-covered surfaces were lower in HU compared to both other groups at the proximal tibia, no differences at the femoral neck, higher in HU + Ir compared to both other groups at L4, and lower in Con compared to both HU groups at the proximal humerus. d Representative images of osteoclast-covered surfaces and osteoid covered surfaces. Error bars = standard deviation. Groups not sharing the same letter are statistically different within that bone site (from Tukey HSD; p < 0.05). No letters within a site indicates no statistical differences.

Fig. 2. Cancellous bone formation rate of the proximal tibia metaphysis (PT), femoral neck (FN), 4th lumbar vertebrae (L4), and proximal humerus (PH).

a At the proximal tibia, BFR was lower in HU vs. both Con and HU + Ir with HU + Ir also lower than Con. At the femoral neck, HU + Ir had higher BFR than both Con and HU. At L4, HU was lower than Con with HU + Ir higher than both groups. At the proximal humerus, Con was lower than HU + Ir with HU not different from either group. b Mineralized surface at the proximal tibia was lower in HU compared to both other groups with higher mineralized surface in HU + Ir compared to Con and HU at the femoral neck. At L4, mineralized surface was lower in HU compared to Con with HU + Ir higher than both HU and Con. There were not statistical differences in mineralized surface at the proximal humerus. c At the proximal humerus, mineral apposition rate was lower in HU compared to Con and HU + Ir with HU + Ir higher than HU alone. There were not differences at the femoral neck. At L4, mineral apposition rate was lower in HU vs. both other groups and in the proximal humerus mineral apposition rate was lower in Con vs. both HU groups. d Representative image of single and double label in dynamic histomorphometry for bone formation rate. Error bars = standard deviation. Groups not sharing the same letter are statistically different within that bone site (from Tukey HSD; p < 0.05). No letters within a site indicates no statistical differences.

Fig. 3. Immunohistochemistry for cathepsin-K-covered bone surfaces, a surrogate of osteoclast surfaces, at the distal femur and proximal humerus.

a) %Cathepsin-K-positive surfaces were higher in the distal femur of HU rats vs. both other groups. In the proximal humerus, Con had the highest %Cathepsin-K-positive surfaces followed by HU and then HU + Ir. Error bars = standard deviation. Groups not sharing the same letter are statistically different within that bone site (from Tukey HSD; p < 0.05). b) Representative histological images of cathepsin-K staining in cancellous bone.

Fig. 4. Immunohistochemistry of osteocyte proteins in the cancellous bone of the distal femur and proximal humerus.

a %TNF-α-positive osteocytes were higher in HU compared to both Con and HU + Ir with HU + Ir higher than Con. At the proximal humerus, Con had higher %TNF-α-positive osteocytes compared to both HU groups. b %IL-17-positive osteocytes were higher in HU vs. Con and HU + Ir at the distal femur and higher in Con vs. both HU groups at the proximal humerus. c %RANKL-positive osteocytes were higher in HU vs. Con and HU + Ir with HU + Ir higher than Con at the distal femur. At the proximal humerus, Con was higher than HU and HU was higher than HU + Ir. d There were not statistical differences in %OPG-positive osteocytes at either bone site. e %Sclerostin-positive osteocytes were higher in HU vs. Con and HU + Ir with HU + Ir higher than Con at the distal femur. At the proximal humerus, %sclerostin-positive osteocytes were higher in Con vs. both HU groups. f Representative image of osteocyte immunohistochemistry. Error bars = standard deviation. Groups not sharing the same letter are statistically different within that bone site (from Tukey HSD; p < 0.05).