Functional folate receptor cell-associated inflammatory cytokines predict the progression of knee osteoarthritis

Abstract

Purpose: In our prior study using 99mTc-EC20 (Etarfolatide) imaging, we observed folate receptor positive (FR+) cells in vivo in the synovium of the majority (76%) of knees of patients with osteoarthritis (OA). The quantity of these FR+ cells was associated with radiographic severity of knee joint space narrowing and osteophyte and knee pain. To identify the cells with functional folate receptors and their involvement in disease pathogenesis and progression in knee OA, we characterized the immune cell populations of OA synovium and synovial fluid by flow cytometry and evaluated the correlation of functional folate receptor positive phenotypes with cytokine production, knee OA severity and progression. Based on results from our pilot Etarfolatide study, we hypothesized that immune cells bearing functional folate receptors would be pro-inflammatory. Methods: For these analyses, we evaluated specimens from three different cohorts. Total Knee Replacement (TKR) Cohort: waste synovial tissue (SV) and matched synovial fluid (SF) obtained from knee OA patients at the time of total knee arthroplasty (n=18) at Duke University Hospital. Cell populations from SV and SF were phenotyped for cell surface markers and functional FR (FR+ detected by incubating cells with fluorescently labeled folate conjugate) using polychromatic flow cytometry. The EC20 Scan Cohort: SF from radiographic knee OA patients (Kellgren-Lawrence (KL) grade 1-4) (n=26 knees), Etarfolatide and computed tomography (CT) imaging. The Prediction of Osteoarthritis Progression (POP) Cohort: SF samples from radiographic knee OA patients (KL grade 1-3) with three-year follow-up and available SF (n=107 knees). Inflammatory cytokines were quantified in SF from all three cohorts and concentrations correlated with the immunophenotypic and imaging data. Results: Using polychromatic flow cytometry, FR+ macrophages and neutrophils were identified in both the SF and SV of the TKR cohort (Table 1). On a per cell basis, neutrophils took up significantly more folate conjugate than macrophages. Among the SF cytokines, transforming growth factor beta 1 (TGF-β1) and neutrophil elastase correlated with FR+ macrophages and FR+ neutrophils, respectively. The SF concentration of both of these cytokines were strongly correlated with the in vivo FR+ synovial phenotype by Etarfolatide uptake in the EC20 cohort (p < 0.0001). TGF-β1 and elastase were both strongly correlated with the osteophyte (OST) score (p < 0.05) in the EC20 (evaluated by CT) and POP (evaluated by radiograph) cohorts. Lastly, in the POP cohort, baseline TGF-β1 and elastase were associated with knee OA progression based on disease outcomes (no progression, radiographic features of OST, OST plus joint space narrowing (JSN) and total knee replacement (TKR) in sequence for linear trend analysis) (p<0.0001 and p=0.0014, respectively) (Fig 1A). Receiver operating characteristics (ROC) analysis of SF TGF-β1 (adjusting for age, BMI, gender, baseline OST and JSN) for the determination of any knee OA progression (either OST, JSN or TKR progression vs. non-progression) yielded an area under the curve (AUC) of 0.95 (95% CI [0.87, 0.98]) (Fig 1B). ROC analysis of elastase yielded an AUC of 0.90 (CI [0.80, 0.95]) (Fig 1B). Applying the Youden index as the cut-off value, for TGF-β1 the sensitivity was 97% (CI [89, 99]), the specificity 85% (CI [62, 97]), and the positive predictive value (PPV) 95% (CI [88, 98]). For elastase the sensitivity was 85% (CI [74, 0.92]), the specificity 86% (CI [65, 97]), and the PPV 95% (CI [86, 98]). Conclusions: We have identified activated macrophages and neutrophils as the functional folate receptor bearing cells in knee OA. The correlation of SF TGF-β1 and elastase with the EC20 imaging phenotype confirmed the pro-inflammatory nature of the FR+ cells in vivo. We further confirmed the pro-inflammatory nature of these cells based on the association of these cell markers with knee OA progression. Taken together, these results suggest that these cells could be potential therapeutic targets in OA. The promising AUC scores for these markers also suggest that SF TGF-β1 and elastase could be the novel biomarkers of risk of progression in knee OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)