Effects of WNT5A signaling-mediated inflammation of macrophages upon cartilage homeostasis

Abstract

Purpose: To construct and comparehumanbone marrow-derived mesenchymal stem cells (MSCs) two dimension (2D)and bone marrow aspirates in vitrtranswell separated co-cultural models with recombinant adeno-associated virus (rAAV)-Wnt5a-modified activated macrophages and investigate the effect of Wnt5a signaling upon cartilage homeostasis through the regulation of macrophage pro-inflammatory responses. Methods: Macrophages, MSCs,and bone marrow aspirates specimens were extracted from 6 patients with severe knee deformities undergoing total knee arthroplasty (2 males, 4 females, 58-71 years old). The synovial tissues of knee joints were exposed to type II collagenase and obtained single cell suspensions, and the purity of macrophages was determined by Ficoll gradient centrifugation and anti-CD14 antibody flow cytometry. The macrophages were transduced with rAAV-lacZ or Wnt5a respectively for 48 hours prior to the stimulation of IFN-γ combined TNF-α for 24 hours, and subsequently transferred to the upper compartment of the 96-well transwell plate, with hMSCs 2D monolayer, and bone marrow aspirates at the lower compartment, respectively (Figure 1 & 2). Cell morphology and proliferation (cellularity), viral transfection efficiency and secretion of proinflammatory cytokine were estimated by Haemotoxylin and Eosin (H&E) staining, toluidine blue staining, X-gal staining, anti-Wnt5a, anti-II and X collagen immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) assay, respectively. Results: The isolated synovial macrophages were positive against anti-CD14 primary antibody, with a percentage of 90.31%, indicating that the cells were macrophages. The level of monocyte chemotactic protein-1 in supernatants was significantly increased after stimulation with IFN-γ and TNF-α, indicating that the macrophages were activated and at proinflammatory condition (Figure 3). After transduction with rAAV-lacZ for 3 days, X-gal staining indicated that lacZ gene transfer could efficiently transducethe activated macrophages, with the efficiency over 97.50% for at least 21 days. rAAV-Wnt5a-modified macrophages enhanced the intrinsic expression of Wnt5a, stimulated the extrinsic expression of Wnt5a in the above two models and inhibited the secretion of MCP-1. The expression of MCP-1 in Wnt5a group was 14.76 and 61.51 pg/ml, respectively (F = 6.78 and F = 4.67, p<0.05 or 0.01). In addition, rAAV-Wnt5a gene transfer could promote cell proliferation (F = 42.15 and F = 99.38, p<0.05 or 0.01) and chondrogenic differentiation and cartilage matrix synthesis,with a statistical significance in toluidine blue (F = 5.05, p<0.05 or 0.01) and immunohistochemical staining against type II and type X collagen (F = 20.65 and 26.47, p<0.05 or 0.01). In the 2D single-layer transwell co-culture model, the cell density of the Wnt5a group was 37.75 / μm2, with 2.40, 2.19 and 2.16 times comparing the remaining 3 groups respectively (Figure 4), while the cell proliferation capacity of the bone marrow aspirates transwell co-culture model in Wnt5a group was significantly enhanced to 99.28/ μm2, with 1.59, 1.73 and 1.91 times comparing the remaining 3 groups respectively. Moreover, Wnt5a transfection also promotes the expression of type II and Type X collagen in the bone marrow aspirates transwell co-culture model, with semi-quantitative analysis of 93.50 and 61.50, respectively, with 1.45, 1.56, 1.41 and 3.24, 3.08, 2.80 times comparing to lacZ and lacZ + KN93 and Wnt5a + KN93 group respectively (Figure 5). Conclusions: Under the condition of macrophage and MSCs 2D monolayer or aspirates transwell co-culture, rAAV-mediated overexpression of Wnt5a can promote the maintenance of cartilage homeostasis and chondrogenic differentiation of MSCs via macrophage inflammatory response,macrophages may affect cartilage homeostasis and MSCs chondrogenesis through Wnt5a signaling pathway.Fig. 2View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5View Large Image Figure ViewerDownload Hi-res image Download (PPT)