1785: Soluble CD14 Amplifies Chondrocyte Inflammatory Responses to Lipopolysaccharide and Is Targetable by Anti-CD14 Therapy

Background/Purpose: Osteoarthritis (OA) is characterized by a pro-inflammatory joint microenvironment, including elevated levels of endogenous pattern recognition receptor ligands in synovial fluid. Notably, both lipopolysaccharide (LPS) and its soluble co-receptor, soluble CD14 (sCD14), are increased in OA synovial fluid, and sCD14 levels correlate with disease severity. sCD14 facilitates LPS transfer to Toll-like receptor 4 (TLR4), potentiating immune responses to otherwise sub-threshold ligand levels. Although this mechanism has been demonstrated in myeloid cells and synovial fibroblasts, whether sCD14 can enhance LPS-driven inflammatory activation of human chondrocytes, at the low levels found in OA joints, remains unclear.

Methods: Primary human articular chondrocytes (hACs) were isolated from non-arthritic knee cartilage (obtained through the National Disease Research Interchange) and treated with LPS at OA-relevant (1 ng/mL) or supraphysiologic (100 ng/mL) concentrations, with or without sCD14 (2 µg/mL). NF-κB activation was assessed via Western blot, inflammatory gene expression by qPCR, and IL-8 and sCD14 secretion by ELISA (n=2–4). To assess CD14 dependency of the responses, C28/I2 chondrocytes (Merck) were treated with anti-CD14 antibody in parallel experiments.

Results: Disease-relevant concentrations of LPS (1 ng/mL) alone did not increase IL-8 secretion by hAC; however, co-treatment with sCD14 significantly elevated IL-8 protein levels compared to either condition alone (Fig. 1A). Expression of IL-8, CCL5, and CXCL10 was moderately increased with 1 ng/ml LPS, with trends toward enhancement by sCD14. In contrast, supraphysiologic LPS (100 ng/mL) induced robust inflammatory gene expression that was not further enhanced by sCD14, indicating a threshold effect (Fig. 1B-D). NF-κB activation was modestly affected by sCD14, suggesting involvement of additional signaling pathways (Fig. 1E, F). We further observed that chondrocytes did not secrete sCD14 under basal or LPS-stimulated conditions, supporting an exogenous origin of sCD14 (Fig. 2). Notably, sCD14 alone induced low-level inflammatory gene expression, suggesting it may function as a damage-associated molecular pattern in chondrocytes.

In a therapeutic approach, CD14 blockade abrogated NF-κB activation induced by high-dose LPS, regardless of sCD14 presence, but had no effect on IL-8 expression. Importantly, at the OA-relevant concentration of 1 ng/mL LPS, CD14 inhibition reversed the sCD14-mediated increase in IL-8, effectively dampening inflammation (Fig. 3A, B).

Conclusion: sCD14 enhances chondrocyte sensitivity to disease-relevant concentrations of LPS, promoting inflammatory activation in a CD14-dependent manner. These findings identify sCD14 as a potential amplifier of chondrocyte-inflammatory responses in OA and support the potential of CD14 blockade to mitigate low-grade inflammation in cartilage.