Interleukin 6, RANKL, and Osteoprotegerin Expression by Chikungunya Virus-Infected Human Osteoblasts

TO THE EDITOR—Chow et al [1] recently implicated interleukin 6 (IL-6) in the persistent arthralgia that occurs in some patients following infection with chikungunya virus (CHIKV). They observed that plasma IL-6 concentrations in patients with persistent arthralgia were higher than those in fully recovered patients; the significance of this observation is supported by the known role of IL-6 in causing joint pain [2]. A role for IL-6 in persistent arthralgia is further supported by the finding by Hoarau et al [3] that IL-6 is specifically expressed in the affected joint during chronic chikungunya disease. Nevertheless, the plasma IL-6 concentration in patients with chronic disease is low (interquartile range, 4–40 pg/mL) and close to normal values [1]. IL-6 is expressed by a variety of cell types, including osteoblasts, and the low circulating levels suggest the joint as a potential source of this cytokine. IL-6 stimulates the release of RANKL [4] and inhibits the one of its decoy receptor osteoprotegerin (OPG) [5] by osteoblasts, therefore promoting osteoclastogenesis and bone resorption [6]. The RANKL/ OPG ratio indeed drives osteoclastogenesis and osteoclast activation [7]. This raises the possibility that dysregulation of IL-6, RANKL, and OPG during CHIKV infection may contribute to joint pathology. In this context, we aimed to determine whether osteoblasts may be involved in CHIKV induced chronic rheumatic syndromes. First, we tested whether primary human osteoblasts are susceptible to CHIKV infection in vitro. Osteoblast cultures were prepared from bone samples obtained from a healthy male subject during a knee operation for a cause unrelated to arthritis. The patient’s medical history indicated no autoimmune disorders, metabolic diseases, intake of immune suppressant/stimulating drugs, or immunotherapy for 3 months before surgery. Bone fragments were cultured in α minimum essential medium supplemented with 10% fetal calf serum, 100 mM ascorbic acid, 20 mM HEPES, and 2 mM L-glutamine. After 2 weeks, confluent cells surrounding fragments were collected and subcultured. Osteoblast characterization by osteocalcin staining and measurement of alkaline phosphatase activity showed >98% purity [8]. Cell monolayers were infected with CHIKV at a multiplicity of infection of 0.1 for 1 hour at 37°C, washed, and fed with fresh media. As shown in Figure 1A, CHIKV replicated in osteoblasts between days 1 and 20 after infection, at levels comparable to that described for macrophages [9]. The decrease in virus production with