ALPHA defensin (human neutrophil peptide-1) slows surgically-induced osteoarthritis in association with promoting M1 to M2 macrophage polarization

Purpose: Emerging evidence suggests that macrophages play an important part in the low-grade inflammation implicated in the pathogenesis of osteoarthritis (OA). α-defensins are a group of low molecular weight molecules, released from apoptotic human neutrophils, which augment the antimicrobial capacity of macrophages while also having a significant ability to inhibit the biosynthesis of proinflammatory cytokines. In the current study, we evaluated the in vitro and in vivo effects of α-defensin (Human Neutrophil Peptide-1, HNP-1) on macrophage polarization and OA. Methods: Synovial tissue was biopsied and synovial fluid was collected from 6 patients with Kellgren-Lawrence (KL) grade 3-4 knee OA. Synovitis was scored on haematoxylin and eosin (H&E) stained slides. The abundance of M0, M1 and M2 macrophage was evaluated by synovial tissue immunofluorescence (IF) using CD68, CD16 and CD206, respectively. The proportion of M1 and M2 macrophages isolated from synovial fluid and synovium was also qualified by flow cytometry using CD68+/CD16+ and CD68+/CD206+, respectively. We evaluated the ability of different concentrations of α-defensin to polarize THP-1 activated monocytes from M1 to M2 macrophages in vitro. These α-defensin stimulated macrophages were cocultured with OA chondrocytes followed by evaluation of chondrocyte gene and protein expression by RT-qPCR and western blot for Col2a1, Acan, Mmp3, Mmp13, Adamts4 and Adamts5. To test α-defensin in vivo, 24 Wistar rats were divided into three groups; sham or surgical destabilization of the meniscus (DMM) was followed one week later by intra-articular injection (250 μL) weekly for 8 weeks: Group 1-Sham+/Saline+ , Group 2-DMM+/α-defensin+ (10ng/mL*250μL), Group 3-DMM+/Saline+. Nine weeks after the surgery, X-ray and μCT were performed, joints were evaluated for OA and synovitis on safranin O and H&E stained central sections, respectively. Data were analyzed using one-way ANOVA. Results: By immunofluorescence, synovial M1 macrophages were more abundant than M2 (456.5±28.7 vs. 125.3±47.9, p<0.001, Figure 1). By flow cytometry, M1 macrophages (CD68+/CD16+) were also more abundant than M2 (CD68+/CD206+) in both synovial tissue (24±1.3% vs. 2.9±1.0%, p<0.001) and OA synovial fluid (13.4±1.0% vs. 10.4±1.2%, p=0.03). Optimal in vitro polarization of M1 to M2 macrophages was achieved by α-defensin 10 ng/mL for 48 hours yielding a significant increase in IL-10, CCL18 and CCL22 and a significant decrease in TNF-α and CCL3 in gene expression. When macrophages were incubated in vitro with α-defensin, increased M2 polarization was confirmed by an increase in the M2 population (change in CD11b+/CD16+ cells from 57.5±1.9% to 12.3±0.7%). Compared to OA chondrocytes cocultured with non-polarized macrophages, OA chondrocytes cocultured with α-defensin polarized macrophages showed significantly increased Col2a and Acan and significantly decreased Mmp3, Adamts4 and Adamts5 gene expression; expression results were confirmed by western-blot. Macroscopic grading demonstrated better cartilage morphology of rats injected with α-defensin. Both x-ray and μCT showed osteophyte formation only in rats of the DMM+/α-defensin+ group. Both OARSI score and synovitis scorez were lower in the DMM+/α-defensin+ group compared to the DMM+/Saline+ group. Conclusions: Alpha-defensin can promote M1 to M2 macrophage polarization in vitro, can promote beneficial effects on chondrocytes indirectly via M2 macrophage polarization, and attenuate severity of OA in vivo. Immunomodulation via α-defensin appears to have indirect chondroprotetive effects that warrant further research.