Identification of biomarkers indicative of synovitis in the mouse articular fracture model of PTA

Abstract

Purpose: Post-traumatic arthritis (PTA) can occur rapidly after moderate to severe articular injuries; however, not every patient will go on to develop this condition, and the mechanisms of disease initiation and progression are not fully understood. One of the features of an acute injury is the development of synovitis, characterized by cellular infiltration following injury and resulting in inflammation. Our murine articular fracture model provides an opportunity to study the pathogenesis of OA following joint injury, and to identify biomarkers predictive of PTA development. In this model, C57BL/6 mice develop PTA, whereas the MRL/MpJ strain is protected. Interestingly, although total joint synovial cellular infiltration was not different between strains, the C57BL/6 mice had markedly more synovial macrophages compared to the MRL/MpJ strain. The objective of this study was to identify differences in serum biomarkers during the acute phase of joint pathology in both strains of mice and assess their utility as indicators of pathological features of disease following articular fracture. Methods: All animal procedures were performed in accordance with an IACUC-approved protocol. Male C57BL/6 and MRL/MpJ mice (n=83) were subjected to an articular fracture at 16 weeks of age using an established model. Six mice from each strain did not receive a fracture and served as pre-fracture controls. Mice were sacrificed at 0, 1, 7, 14, and 56 days after fracture (n=6-11 per strain per time point) at which time blood was collected. The left (fractured; Fx) and right (non-fractured; Non-Fx) limbs were harvested. Histology sections (FFPE, 8μm thick in coronal plane) of all limbs were evaluated by three blinded graders for synovial inflammation at the synovial insertion of the lateral tibia, lateral femur, medial tibia, and medial femur and summed, using a modified form of an established standardized, semi-quantitative synovitis score. ELISA assays were used to quantify serum biomarkers including pro-inflammatory cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, KC, IL-10, IL-12p70, TNFα), MMP-3, TIMP-1, and VCAM-1. Non-parametric analyses were performed to assess differences in synovitis between Non-Fx and Fx limbs as well as differences in synovitis and biomarkers between strains. Spearman correlations were performed to assess the association of biomarkers with synovitis scores. Results: Articular fracture was associated with an increase in synovial inflammation in both mouse strains. Compared to the non-fractured contralateral limb at baseline, there were significant increases in synovitis in the fractured limb in the C57BL/6 mice at 7, 14, and 56 days post-fracture, but only at 14 days post-fracture (p<0.05) for the MRL/MpJ strain (Figure 1). Serum concentrations of multiple biomarkers were significantly higher in the C57BL/6 strain: KC (14 and 56 days), MMP-3 (4 hours and 7 days), and VCAM-1 (14 and 56 days). In contrast, serum IL-6 and TIMP-1 (1, 14, and 56 days) were higher post-Fx in the MRL/MpJ strain. Compared to the MRL/MpJ strain, the ratio of MMP-3 to TIMP-1 was significantly higher in the C57BL/6 strain at 1 (p=0.03), 7 (p=0.004), 14 (p=0.002), and 56 days post-Fx (p=0.0008), indicating higher levels of degradation in this strain. Synovitis in the fractured limb was significantly positively correlated with IL-1β (rs=0.35; p=0.01), IL-6 (rs=0.29; p=0.04), MMP-3 (rs=0.54; p<0.0001), and TIMP-1 (rs=0.35; p=0.01), and significantly negatively correlated with CXCL1/KC (rs=-0.31; p=0.03), an IL-8 functional homologue. Interestingly, the MMP-3/TIMP-1 ratio was significantly correlated with synovitis of the fractured limb of the C57BL/6 strain (p=0.004). Conclusions: It has become clear that the tissues of the entire joint organ contribute to OA development and contribute to PTA development. Synovitis occurs acutely after injury in both the PTA-susceptible (C57BL/6) and PTA-resistant (MRL/MpJ) strains. Whereas the histological synovitis scoring was unable to distinguish differences between strains, serum analyses revealed higher levels of multiple inflammatory biomarkers and mediators of joint degradation in the PTA-susceptible, C57BL/6 strain. Moreover, the MMP-3/TIMP-1 ratio was strongly correlated with synovitis in the C57BL/6 strain, indicating its ability to identify a degradative synovial phenotype. These biomarkers should be useful for monitoring the destructive and inflammatory responses that result in the development of OA.