Osteoblasts from osteoarthritis patients show enhanced susceptibility to Ross River virus infection associated with delayed type I interferon responses

Abstract

Background: Arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) have caused widespread outbreaks of chronic polyarthritis. The inflammatory responses in alphavirus-induced arthritis and osteoarthritis (OA) share many similar features, which suggests the possibility of exacerbated alphavirus-induced bone pathology in individuals with pre-existing OA. Here, we investigated the susceptibility of osteoblasts (OBs) from OA patients to RRV infection and dissected the immune mechanisms elicited from infection.

Methods: Primary hOBs obtained from trabecular bone of healthy donors and OA patients were infected with RRV. Infectivity and viral replication were determined using flow cytometry and plaque assay, respectively. Real-time PCR was performed to determine expression kinetics of type I interferon (IFN)-related immune mediators and osteotropic factors.

Results: OA hOBs showed enhanced RRV infectivity and replication during infection, which was associated with delayed induction of IFN-β and RIG-I expression. Enhanced susceptibility of OA hOBs to RRV was associated with a more pronounced increase in RANKL/OPG ratio and expression of osteotropic factors (IL-6, IL-1β, TNF-α and CCL2) in comparison to RRV-infected healthy hOBs.

Conclusions: Delayed activation of type I IFN-signalling pathway may have contributed to enhanced susceptibility to RRV infection in hOBs from OA patients. RRV-induced increases in RANKL/OPG ratio and expression of osteotropic factors that favour bone resorption, which may be exacerbated during osteoarthritis. This study provides the novel insight that osteoarthritis may be a risk factor for exacerbated arthritogenic alphaviral infection.

Figure 1

Enhanced RRV replication in primary hOBs of OA patients. (A) Phenotypic characterization of primary hOBs. Primary hOBs were fixed and stained for osteocalcin (green) and nuclei (blue), and visualized by confocal microscopy. Magnification, ×20. Images are representative of 2 independent experiments. Primary hOBs were infected with RRV-EGFP at MOI 1 and cells and supernatant were harvested at different time points. (B) Dose-dependent infection of hOBs. Cell harvested at 24 hpi were fixed, stained for 7-AAD, and subjected to FACS analysis. FACS scatter plots are representative of the data obtained for 3 independent experiments. (C) Quantification of percentage EGFP⁺ cells after infection. (D) Supernatant was harvested and RRV titre was determined by plaque assay on Vero cells. Data (n =4) are presented as mean ± SEM. *P <0.05, two-way ANOVA, Bonferroni post-test.

Figure 2

Delayed IFN-signalling pathway in primary hOBs of OA patients during RRV infection. Primary hOBs were infected with EGFP-RRV at MOI 1 and cells were harvested at different time points for RNA extraction. Transcriptional profiles of (A) IFN-β and (B) RIG-I were determined using qRT-PCR. Data are normalized to HPRT and shown as fold expression relative to healthy mock-infected group. Data (n =4) are presented as mean ± SEM. *P <0.05, one-way ANOVA, Tukey’s post-test.

Figure 3

Underlying OA condition further perturbs RANKL/OPG ratio after RRV infection in primary hOBs. Primary hOBs were infected with EGFP-RRV at MOI 1 and cells were harvested at different time points for RNA extraction. Transcriptional profiles of (A) RANKL and (B) OPG were determined using qRT-PCR. (C) RANKL/OPG ratio is shown. Data are normalized to HPRT and shown as fold expression relative to healthy mock-infected group. Data (n =4) are presented as mean ± SEM. *P <0.05, one-way ANOVA, Tukey’s post-test.

Figure 4

Enhanced expression of osteotropic factors in primary hOBs of OA patients after RRV infection. Primary hOBs were infected with EGFP-RRV at MOI 1 and cells were harvested at different time points for RNA extraction. Transcriptional profiles of osteotropic factors (A) IL-6, (B) IL-1β, (C) TNF-α and (D) CCL2 were determined using qRT-PCR. Data are normalized to HPRT and shown as fold expression relative to healthy mock-infected group. Data (n =4) are presented as mean ± SEM. *P <0.05, one-way ANOVA, Tukey’s post-test.