Knockdown of toll-like receptor 4 signaling pathways ameliorate bone graft rejection in a mouse model of allograft transplantation.

Non-union occurring in structural bone grafting is a major problem in allograft transplantation because of impaired interaction between the host and graft tissue. Activated toll-like receptor (TLR) induces inflammatory cytokines and chemokines and triggers cell-mediated immune responses. The TLR-mediated signal pathway is important for mediating allograft rejection. We evaluated the effects of local knockdown of the TLR4 signaling pathway in a mouse segmental femoral graft model. Allografts were coated with freeze-dried lentiviral vectors that encoded TLR4 and myeloid differentiation primary response gene 88 (MyD88) short-hairpin RNA (shRNA), which were individually transplanted into the mice. They were assessed morphologically, radiographically, and histologically for tissue remodeling. Union occurred in autografted but not in allografted mice at the graft and host junctions after 4 weeks. TLR4 and MyD88 expression was up-regulated in allografted mice. TLR4 and MyD88 shRNAs inhibited TLR4 and MyD88 expression, which led to better union in the grafted sites. More regulatory T-cells in the draining lymph nodes suggested inflammation suppression. Local inhibition of TLR4 and MyD88 might reduce immune responses and ameliorate allograft rejection.

Amelioration of Experimental Autoimmune Arthritis Through Targeting of Synovial Fibroblasts by Intraarticular Delivery of MicroRNAs 140-3p and 140-5p.

OBJECTIVE: Synovial fibroblasts (SFs) with aberrant expression of microRNAs (miRNAs) are critical pathogenic regulators in rheumatoid arthritis (RA), and studies analyzing the effect of overexpressing or silencing miRNA expression in arthritis models can contribute to the development of miRNA-based therapeutic strategies. This study was undertaken to examine the hypothesis that miRNAs 140-3p and 140-5p are involved in the pathogenesis of RA, and to determine whether targeting SFs through the intraarticular (IA) delivery of these molecules could ameliorate autoimmune arthritis in mice. METHODS: Synovial tissue samples were obtained from patients with RA. In addition, 2 experimental models in mice were used, collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA). Overexpression of miRNAs 140-3p and 140-5p in SFs and synovial tissue was induced using lentivirus (LV)-mediated transfer of pre-miR-140 precursor molecules. RESULTS: Lower expression levels of miR-140-3p and miR-140-5p were detected in synovial tissue and SFs from patients with RA and from mice in both arthritis models. In mice with CIA and mice with CAIA, the LV-mediated IA transfer of miR-140-3p and miR-140-5p ameliorated arthritis, as determined by clinical examination and histopathologic evaluations showing a decrease in SF densities. Overexpression of miRNAs 140-3p and 140-5p caused a reduction in expression, with correlated kinetic patterns, of their corresponding target molecules sirtuin 1 and stromal cell-derived factor 1 in the SFs and joints of mice. Transfection of miR-140-3p and miR-140-5p into SFs increased cell apoptosis, reduced proliferation responses and migration abilities, and verified the concept that miR-140 expression is regulated by proinflammatory cytokines. CONCLUSION: These results demonstrate that targeting SFs by IA delivery of miRNAs 140-3p and 140-5p can ameliorate autoimmune arthritis. These findings might facilitate the pharmacologic development of molecular-based therapies in RA.