Impact of Tacrolimus in the epithelial-mesenchymal transition in chronic lung allograft dysfonction

Introduction: Bronchiolitis obliterans syndrome (BOS) is characterized by an abnormal remodeling of the bronchial epithelium causing fibrotic obliteration and occurring in 50% of lung transplanted patients after 5 years. One of the mechanisms involved is the epithelial-mesenchymal transition (EMT). Microparticles (MPs) are fragments of plasma membrane emitted by cell in response to stress and putative cellular effectors of BOS. The impact of immunosuppression and inflammatory pulmonary status remain debated. We aimed at assessing in vitro effect of Tacrolimus (TC), calcineurin inhibitor, and MPs on bronchial epithelial cells. Methods: BEAS-2B, bronchial epithelial cells were submitted to TC (10µM) and/or MPs (30nM) for 72h. Pro-inflammatory MPs were harvested from LPS of Pseudomonas aeruginosa-stimulated monocytes THP1, washed et concentrated. Cells were used to test TC and MPs. Cellular morphology was analyzed by optical microscopy, epithelial and mesenchymal markers by Western blot and migratory cell capacity by scratch-test. Results: Cells treated by TC, MPs or both stimuli, had modified cellular morphology and a mesenchymal profile with a significant up-regulation in vimentin (TC: 26.73 ± 0.585 vs untreated: 1.00, p = 0.005) and a significant down-regulation in E-cadherin (TC: 0.3234 ± 0.005 vs. untreated: 1.00, p <0.0001). Combined treatment by TC and MPs led to a drastic enhancement in migration patenting as compared to TC or MPs alone (gap filling 71%, 50% and 51% respectively). Conclusion:In vitro, TC combined to pro-inflammatory MPs lead to favor exaggerated EMT. Immunosuppression regimens should take into account the inflammatory status of lung transplanted patients.