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

To investigate if underlying OA has any effect on viral replication during RRV infection, primary hOBs cultured from trabecular bones of healthy donors and OA patients were used. The cultured primary hOBs were phenotypically characterized using osteocalcin, an OB marker, prior to RRV infection (Figure 1A). To determine the infectivity of these primary hOB cultures, they were infected with RRV-EGFP at a series of multiplicity of infection (MOI) from 1 to 10. Infectivity of both healthy and OA hOBs was demonstrated in a dose-dependent manner (Figure 1B). The kinetics of RRV replication in the primary hOBs was further determined through a 96 h time-course infection, with cells and supernatant harvested at 24, 48 and 96 hours post-infection (hpi) for FACS analysis and plaque assays. Peak RRV replication was observed at 24 hpi for both healthy and OA hOBs, with a subsequent decline over the next 72 h. Interestingly, FACS analysis of the harvested cells revealed significantly enhanced RRV infectivity in OA hOBs at 24, 48 and 96 hpi (Figure 1C). Consistent with the enhanced infectivity in OA hOBs, higher levels of virus were also recovered from the supernatant of infected OA hOBs at 24 and 48 hpi, compared to healthy hOBs (Figure 1D).

The type I IFN signalling pathway serves as the first line of host defence against pathogen invasion. We sought to determine the involvement of type I IFN in the contribution of enhanced RRV infectivity and replication in OA hOBs. Infected primary hOBs were harvested at 24, 48 and 96 hpi for determination of IFN-β and RIG-I mRNA expression (Figure 2). At the peak viral replication of 24 hpi, RRV-infected OA hOBs had significantly lower transcriptional induction of both IFN-β and RIG-I compared to RRV-infected healthy hOBs. The induction of IFN-β and RIG-I in RRV-infected OA hOBs gradually overtook that of RRV-infected healthy hOBs at 48 and 96 hpi, respectively. Together, these data suggest that enhanced susceptibility and viral replication in OA hOBs may be modulated through delayed induction of type I IFN.

Alphavirus-induced arthritis and OA are inflammatory arthritides that have been associated with bone disorders [17],[22]. Recently, we demonstrated that RRV infection can elicit bone loss through perturbed OB function. Therefore, we investigated the effect of RRV infection on bone regulatory function of OA hOBs by measuring RANKL and OPG, which are the fundamental elements of bone homeostasis. Briefly, primary hOB cultures were infected at MOI 1 and harvested at 24, 48 and 96 hpi for determination of RANKL and OPG transcript expression. RANKL expression in OA hOBs was significantly higher at all time points than in healthy hOBs after RRV infection. A drastic increase in RANKL expression of ~20-fold between 24 to 96 hpi was observed in RRV-infected OA hOBs, compared to ~5-fold increase for RRV-infected healthy hOBs. No significant difference in RANKL expression was observed between mock-infected healthy and OA hOBs (Figure 3A). Higher levels of OPG expression were detected in mock-infected OA hOBs compared to mock- or RRV-infected healthy hOBs. After RRV infection, OA hOBs demonstrated higher expression of OPG compared to healthy infected hOBs at all tested time points. No significant changes in OPG expression were observed between mock- or RRV-infected healthy hOBs at all tested time points. However, at 48 and 96 hpi, OPG levels were significantly lower in the RRV-infected OA hOBs compared to mock-infected OA hOBs (Figure 3B). Thus, RRV infection resulted in a markedly elevated RANKL/OPG ratio in both healthy and OA hOBs compared to mock-infected groups. Interestingly, OA hOBs exhibited a significantly higher RANKL/OPG ratio than healthy hOBs after RRV infection at 48 and 96 hpi (Figure 3C), suggesting that underlying OA may exacerbate RRV-induced bone pathology by further increasing the RANKL/OPG ratio.

Pro-inflammatory mediators such as IL-6, IL-1inflammatory mediators such as IL-6, TNF-α and CCL2 contribute to alphaviral disease pathogenesis. Interestingly, these immune mediators also function as osteotropic factors, with a pivotal role in modulating bone remodelling [23],[24]. Transcriptional analysis of these osteotropic factors was performed to assess their expression kinetics. IL-6 expression was up-regulated at all tested time points after RRV infection in both healthy and OA hOBs. At 24 and 48 hpi, RRV infection of OA hOBs elicited consistently lower levels of IL-6 transcription compared to healthy hOBs, but its expression was increased at 96 hpi (Figure 4A). Early induction of IL-1β expression was observed in both healthy and OA hOBs after RRV infection at 24 hpi, which peaked at 48 hpi, with ~30-fold increase for RRV-infected healthy hOBs and ~90-fold increase for RRV-infected OA hOBs. At 96 hpi, the RRV-induced expression of IL-1β in healthy hOBs declined, while transcription continued to increase dramatically to ~600-fold change in RRV-infected OA hOBs (Figure 4B). Elevated expression of TNF-α was detected at 24 hpi in RRV-infected healthy and OA hOBs, but to a lesser extent for the OA group. The induction of TNF-α declined rapidly at 48 hpi and reverted to basal level by 96 hpi (Figure 4C). CCL2 expression was highly up-regulated in healthy hOBs after RRV infection at 24 hpi and decreased gradually over the next 72 h. In contrast, RRV-induced CCL2 expression in OA hOBs only commenced at 96 hpi (Figure 4D). Collectively, the elevated expression of these pro-inflammatory/osteotropic factors at later stages of infection may stimulate RANKL-mediated osteoclastogenesis.

Stocks of RRV strain T48 (RRV-T48) were generated from the full-length T48 cDNA clone [39]. Stocks of RRV that expressed enhanced green fluorescent protein (RRV-EGFP) were generated by inserting a second RRV 26S promoter sequence at the 3' end of the viral genome, followed by the coding sequence for EGFP (kindly provided by Dr Mark Heise, University of North Carolina). All titrations were performed by plaque assay on Vero cells as described previously [40].

Primary human OB (hOB) cultures were obtained from trabecular bone specimens of 7 healthy individuals (4 males, 3 females), aged 50-60 years. Healthy hOBs were obtained from individuals undergoing orthopaedic operations for causes unrelated to arthritis and osteoporosis (such as ligament reconstruction or trauma cases). OA trabecular bone specimens were obtained from femur neck of 5 patients (3 males, 2 females) undergoing primary or revision surgery. The patients ranged in age from 50-60 years and were diagnosed with OA according to the World Health Organisation (WHO) criteria, mainly based on clinical presentation, radiographic findings. All human specimens were collected with the permission of the Human Research Ethics Committee of Australian National University (human ethics approval-ETH-9-07-865) and Australian Capital Territory (ACT) Health, together with informed patient consent. The exclusion criteria for recruiting hOB donors was (i) taking immune suppressant/stimulating drugs, cytokine, or IFN therapy in the 3 months before surgery; (ii) treatment with anabolic agents; and (iii) low quality of the bone fragment, socio-economic or ethic characteristics and age not matching with the healthy group, in the opinion of the investigators, could compromise the study. Briefly, the bone fragments were washed with PBS and cultured in α-MEM (Sigma-Aldrich) supplemented with 10% HI-FCS, 1% antibiotic solution, 100 μM ascorbic acid, 20 mM Hepes, and 2 mM L-glutamine at 37°C with 5% CO₂. After 2 weeks, outgrowing cells surrounding bone fragments were cultured and designated as "passage 1." The hOBs cultured in our laboratory were able to proliferate in vitro for up to five passages but were used at passage 2 in all experiments.

To characterise cultured hOB, osteocalcin expression was determined by staining hOB with primary mouse anti-human osteocalcin monoclonal antibodies (R&D Systems, Minneapolis, USA) and Alexa-Fluor 488 (AF488) goat anti-mouse IgG (Invitrogen, Melbourne, Victoria, Australia), followed by DAPI staining and visualisation using an FV1000 confocal microscope (Olympus, Australia).

Primary hOBs were seeded in 24-well plates at a density of 1 - 10⁵ cells per well overnight. Cells were then infected with RRV-EGFP at a multiplicity of infection (MOI) of 1. After 1 h, cells were washed twice and cultured in fresh α-MEM with 10% FCS at 37°C with 5% CO₂. At appropriate time points, culture medium was collected for plaque assay and cells were harvested for RNA extraction.

Cells were trypsinsed and washed twice in wash buffer (PBS-2% FCS). Cells were then stained with 7-AAD (7-aminoactinomycin) (eBioscience, San Diego, CA) at room temperature for 10 min. Following 7-AAD staining, EGFP expression in RRV-infected cells was analysed using a CyAn ADP flow cytometer and Kaluza software (Beckman Coulter).

Vero cells were seeded into 24-well plates at a density of 1 - 10⁵ cells per well and cultured to confluence. Serially diluted samples (10⁻¹ to 10⁻⁶) were added to cell monolayers and incubated for 1 h at 37°C in a 5% CO₂ incubator. Cells were then overlaid with OPTI-MEM (Invitrogen, Melbourne, Victoria, Australia) containing 3% FCS and 1% agarose (Sigma Aldrich, Sydney, Australia) and incubated for 48 h in a 5% CO₂ incubator. The cells were fixed with 1% formalin and plaques were visualised by staining with 0.1% crystal violet. Virus titres were expressed as plaque forming units per millilitre (PFU/mL) or PFU per gram of tissue.

RNA was prepared from cell pellets using TRIzol (Invitrogen, Melbourne, Victoria, Australia) according to the manufacturer's instructions. Eluted RNA was stored at −80°C. Total RNA was measured by NanoDrop 1000 spectrophotometer (Thermo Scientific). Extracted total RNA (20 ng/μL) was reverse-transcribed using an oligo(dT) primer and reverse transcriptase (Sigma Aldrich, Sydney, Australia), according to the manufacturer's instructions. SYBR® Green Real-time PCR was performed using 50 ng of template cDNA on a CFX96 Touch™ Real-Time PCR System in 96-well plates, using QuantiTect Primer Assay kits (Qiagen, Hilden, Germany) with the following conditions: (i) PCR initial activation step: 95°C for 15 min, 1°Cycle and (ii) 3-step cycling: 94°C for 15's, follow by 55°C for 30's and 72°C for 30's, 40°Cycles. Amplification specificity was evaluated by a melting curve analysis of PCR products. The fold change in messenger RNA (mRNA) expression relative to mock-infected samples for each gene was calculated with the ΔΔCt method. Briefly, ΔΔCt-=ΔΔCt (RRV-infected) - Ct (Mock-infected) with ΔCtΔ= Ct (gene of interest) - Ct (housekeeping gene - HPRT). The fold change for each gene is calculated as 2^-ΔΔCt.

Viral titers and flow cytometry data were statistically evaluated using two-way analysis of variance (ANOVA) with Bonferroni posttest. Real-time PCR data were analyzed using one-way ANOVA with Tukey's posttest. Differences between groups with P <0.05 were considered significant. All data were assessed for normality using the D'Agostino-Pearson normality test before analysis with these parametric tests. All statistical analyses were performed with GraphPad Prism software, version 5.02.