Significant role for microRNA-21 affecting toll-like receptor pathway in primary graft dysfunction after human lung transplantation

Abstract

MicroRNAs (miRNAs) were recently identified as modulators of immune responses after human lung transplantation (LTx). This study was undertaken to assess the contribution of miRNAs to the pathogenesis of primary graft dysfunction (PGD) after LTx. Of the 39 recipients, 14 (35.9%) developed Grade 3 PGD (i.e., severe PGD) within the first 72 hours of LTx. The remaining 25 recipients (64.1%) had Grade 2 or less PGD, and served as the control group. miRNAs were isolated from cells purified by bronchoalveolar lavage (BAL). Bioinformatic prediction and validation by luciferase reporter assays were performed to identify targets regulated by miR-21. Transfection of human monocytic cell line (THP-1) was conducted to determine miR-21's cellular function. Pilot miRNA profiling of donor BAL samples before implantation in PGD (n = 6) revealed significant upregulation in 44 miRNAs and downregulation in 80 miRNAs compared with control (n = 6). Validation using a separate cohort demonstrated significant underexpression of miR-21 in patients with severe PGD. Furthermore, underexpression of miR-21 levels was negatively correlated with clinical PGD grades (Grade 2 PGD vs Grade 0 PGD: p = 0.042; Grade 3 PGD vs Grade 0 PGD: p = 0.004). Molecular analysis demonstrated that miR-21 targeted key components in the toll-like receptor (TLR) signaling pathway, including TLR4, IRAK3 and CXCL10. Further, incubation of THP-1 cells with cell-free BAL from severe PGD resulted in transactivation of inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). In contrast, increased expression of miR-21 resulted in marked suppression of IL-1-β and TNF-α production. Underexpression of miR-21 may lead to the development of severe PGD by activating key components of the TLR pathway.