Background/Purpose: Soluble CD14, a co-receptor of inflammatory toll-like receptor signaling that is produced primarily by monocytes/macrophages, is present in synovial fluid in patients with OA and positively associates with joint space narrowing and pain. Utilizing both global genetic deletion of CD14 and intra-articular CD14 blockade across mild to severe models of murine PTOA, we aimed to test the hypothesis that CD14 inhibition can attenuate synovial inflammation and associated pain during disease progression.

Methods: DMM: Destabilization of the medial meniscus (DMM) surgery was performed in 12-wk old male C57BL/6 mice. 2) PMX: Partial meniscectomy (PMX) surgery was performed in 12-wk old male mice. 3) HFD-ACLR: Nonsurgical anterior cruciate ligament rupture (ACLR) was performed via overloading in 36-wk old female mice fed a high fat diet (HFD). CD14 knockout: A global genetic knockout of CD14 was utilized and evaluated in the DMM and PMX models. Drug intervention: WT mice were treated intra-articularly (IA) with either an anti-CD14 monoclonal antibody or an IgG2a control (0.5mg/kg). Mice received 3 weekly injections beginning 48 hours post DMM or ACLR injuries. Evoked pain: Knee hyperalgesia was measured using a pressure application measurement (PAM) device. Spontaneous pain behavioral: Spontaneous cage behavior was evaluated using the Laboratory Animal Behavior Observation Registration and Analysis SystemTM. Paw weight bearing distribution and rearing behavior was measured using the Advanced Dynamic Weight Bearing system or a static incapacitance meter system. Single-cell sequencing (scRNA-seq) (n=15): Synovial cells were isolated from DMM- and sham- operated knee joints 4-wks post-surgery. scRNA-seq data was processed using Seurat.

Results: Global deletion of CD14 mitigated knee hyperalgesia in both DMM and PMX models of PTOA, with a significant increase in withdrawal threshold in CD14-KO mice compared to WT post-injury (Fig. 1A-C). Further, delivery of a CD14 blocking antibody mitigated knee hyperalgesia in both DMM and HFD-ACLR models of PTOA (Fig. 1D-F). Spontaneous pain behavior analysis revealed that genetic deletion of CD14 increased mobility (rearing time) in the DMM model, comparing to WT. Further, a protection from weight bearing asymmetry was observed following PMX injury in CD14-KO mice compared to WT (Fig. 1A-C). IA delivery of anti-CD14 increased rearing activity following DMM and protected from weight bearing asymmetry following ACLR injury, both compared to the IgG control (Fig. 1D-F). Mild attenuation of OA histopathology was observed across models. Lastly, synovial scRNA-seq analysis at 4-wks post DMM revealed clear decreases across multiple monocyte populations, as well as a differential modulation of multiple fibroblast populations within the CD14-KO synovium compared to WT (Fig. 2).

Conclusion: The consistent attenuation of pain behavior in both mild and severe models of PTOA strongly supports the potential of targeting CD14 to treat painful OA. Our scRNA-seq results suggest that the protective effects may be mediated in part by decreased inflammatory signaling within the synovium, which influenced both the myeloid and fibroblast populations that emerge after injury.

Figure 1: (A,D) Study schematic. (B,C,E,F) Knee hyperalgesia measured via withdrawal threshold (g). (B,E) Time spent rearing (sec). (C) Weight bearing asymmetry measured as (C) the difference between rear right (DMM) to rear left paw (unoperated) weight (g) or (F) as front to rear paw weight bearing ratio (%/%).

Figure 2: (A) UMAP of cell clusters within WT and CD14 KO synovium harvested 4-wks post injury with re-clustered monocyte populations delineated. (B) Monocyte specific cell cluster breakdown represented as the number and percentage of total cells within monocyte clusters. (C) UMAP of re-clustered monocyte populations. (C) UMAP of cell clusters within WT and CD14 KO synovium harvested 4-wks post injury with fibroblast clusters (Clusters 1, 4, & 9) delineated. (D) Fibroblast cell cluster breakdown represented as the number and percentage of total cells within fibroblast clusters. (pooled n=15/group).

« Back to ACR Convergence 2025

ACR Meeting Abstracts - https://acrabstracts.org/abstract/multi-model-evaluation-of-the-impact-of-cd14-on-synovial-inflammation-and-pain-during-ptoa/