Complement receptor C5aR1 on osteoblasts regulates osteoclastogenesis in experimental postmenopausal osteoporosis

Abstract

In recent years, evidence has accumulated that the complement system, an integral part of innate immunity, may be involved in the regulation of bone homeostasis as well as inflammatory bone loss, for example, in rheumatoid arthritis and periodontitis. Complement may also contribute to osteoporosis development, but investigation of the mechanism is limited. Using mice with a conditional deletion of the complement anaphylatoxin receptor C5aR1, we here demonstrated that C5aR1 in osteoblasts (C5aR1 ^Runx2-Cre mice) or osteoclasts (C5aR1 ^LysM-Cre mice) did not affect physiological bone turnover or age-related bone loss in either sex, as confirmed by micro-computed tomography, histomorphometry, and biomechanical analyses of the bone and by the measurement of bone turnover markers in the blood serum. When female mice were subjected to ovariectomy (OVX), a common model for postmenopausal osteoporosis, significant bone loss was induced in C5aR1 ^fl/fl and C5aR1 ^LysM-Cre mice, as demonstrated by a significantly reduced bone volume fraction, trabecular number and thickness as well as an increased trabecular separation in the trabecular bone compartment. Confirming this, the osteoclast number and the receptor activator of nuclear factor k-B (RANK) ligand (RANKL) serum level were significantly elevated in these mouse lines. By contrast, C5aR1 ^Runx2-Cre mice were protected from bone loss after OVX and the serum RANKL concentration was not increased after OVX. These data suggested that bone cell-specific C5aR1 may be redundant in bone homeostasis regulation under physiological conditions. However, C5aR1 on osteoblasts was crucial for the induction of bone resorption under osteoporotic conditions by stimulating RANKL release, whereas C5aR1 on osteoclasts did not regulate OVX-induced bone loss. Therefore, our results implicate C5aR1 on osteoblasts as a potential target for treating postmenopausal osteoporosis.

Keywords: C5aR1; complement system; osteoblast; osteoclast; ovariectomy.

Figure 1

Bone phenotype of 12-week-old female mice with cell-specific C5aR1 deletion on osteoclasts (C5aR1 ^LysM-Cre) or osteoblasts (C5aR1 ^Runx2-Cre). (A) Cortical thickness (Ct.Th) and (B) flexural rigidity (EI) of the femurs. (C) Representative µCT images of the femurs with the trabecular and cortical compartments. (D) Relative bone volume (BV/TV) of the trabecular bone. (E) Trabecular number (Tb.N). (F) Trabecular thickness (Tb.Th). (G) Trabecular separation (Tb.Sp). (H) Number of osteoblasts (N.Ob/B.Pm) and (I) osteoclasts (N.Oc/B.Pm) per bone perimeter. (J) Concentration of procollagen type I N-terminal propeptide (PINP) and (K) C-terminal telopeptide (CTX-1) in the serum. (L) Representative images of toluidine blue-staining. Arrows indicate osteoblasts. (M) Representative images of tartrate-resistant acid phosphatase (TRAP)-staining. Arrows indicates osteoclasts. Scale bar 50 µm. *p<0.05, n=5–8 per group.

Figure 2

Bone phenotype of C5aR1 ^LysM-Cre and C5aR1 ^Runx2-Cre mice after Sham or OVX. (A) Representative µCT images of the trabecular bone of Sham operated and OVX mice. (B) Uterus weight of Sham operated and OVX mice 8 weeks post-surgery. (C) Flexural rigidity (EI) of the femurs. (D) Tissue mineral density of the cortex (cTMD) and (E) cortical thickness (Ct.Th) of Sham operated and OVX mice. (F) Relative bone volume (BV/TV) of the trabecular bone. (G) Trabecular number (Tb.N). (H) Trabecular thickness (Tb.Th). (I) Trabecular separation (Tb.Sp). *p<0.05, n=6–7 per group.

Figure 3

Cellular parameters in the trabecular bone of the femurs 8 weeks after Sham or OVX. (A) Number of osteoclasts per bone perimeter (N.Oc/B.Pm) and (B) osteoclast surface per bone surface (Oc.S/BS). (C) Number of osteoblasts per bone perimeter (N.Ob/B.Pm) and (D) osteoblast surface per bone surface (Ob.S/BS). (E) Representative images of tartrate-resistant acid phosphatase (TRAP)-staining. Arrows indicate osteoclasts. (F) Representative images of toluidine blue-staining. Arrows indicate osteoblasts. Scale bar 50 µm. *p<0.05, n=5–6 per group.

Figure 4

Serum concentration of bone markers 8 weeks after Sham or OVX. (A) Concentration of the bone markers procollagen type I N-terminal propeptide (PINP) and (B) C-terminal telopeptide (CTX-1) (C) Concentration of receptor activator of nuclear factor-kappa B ligand (RANKL) and (D) osteoprotegerin (OPG). (E) Ratio of RANKL/OPG. *p<0.05, n=5-7 per group.

Figure 5

Quantification and representative immunohistological staining of cytokines in the trabecular bone of the femurs 8 weeks after Sham or OVX. (A) Representative staining of receptor activator of nuclear factor-kappa B ligand (RANKL) and (B) quantitative analysis of RANKL+ cells in the femur. (C) Representative staining of C-X-C motif chemokine 10 (CXCL-10) and (D) quantitative analysis of CXCL-10+ cells in the femur. (E) Representative staining of interleukin 6 (IL-6) and (F) quantitative analysis of IL-6+ cells in the femur. Scale bar 100 µm. *p<0.05, n=4–5 per group.