View Abstract

A2231 - Nuclear Factor of Activated T Cells 2 Contributes to Pulmonary Fibrosis in a Bleomycin Induced Lung Injury Model
Author Block: P. Cao, K. Misumi, N. M. Walker, R. Braeuer, V. N. Lama; Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, MI, United States.
Introduction: Pulmonary fibrosis is a progressive disease with excessive accumulation of extracellular matrix and damage of lung architecture, yet no effective therapy was found. Nuclear factor of activated T cells 2 encodes a member of the NFAT family. We reported recently that NFAT1 promotes lung allograft fibrosis by inducing autotaxin transcription in lung-resident mesenchymal cells. In this study, we aimed to investigate if NFAT1 regulates pulmonary fibrogenesis in a bleomycin induced lung injury model. Methods: NFAT1 knockout mice and control C57BL6 mice were subjected to bleomycin injury by intratracheal injection, and whole lung was harvested for histology analysis, Hydroxyproline assay, Western blot assay and qPCR assay on day 21 post injury. On day 14 post injury, whole lung was digested by Collagenase A and subjected to flow cytometry for analyzing different cell types. Human lung-resident mesenchymal cells were transfected with constitutively active NFAT1 expressing lentivirus with DsRed expressing virus as the control, and the samples was subjected to qPCR analysis. Results: C57BL6 mice with bleomycin injury demonstrated excess collagen accumulation as well as remodeling of lung structure by histology of Trichrome staining. In contrast, NFAT1 knockout mice showed minimal injury and fibrosis after bleomycin injection on day 21. By Hydroxyproline assay, collagen amount increased by ~40% with bleomycin injury in C57BL6 mice, while collagen in NFAT1 knockout mice didn’t increase significantly. Mesenchymal cells from NFAT1 KO mouse demonstrated less autotaxin as well as Collagen I expression under exogenous treatment of lysophosphatidic acid (LPA). Human lung-resident mesenchymal cells expressing constitutively active NFAT1 demonstrated induction of pro-inflammatory cytokines, including IL13, IL8, IL31, CCL5. Fibrogenesis associated genes, including ACTA2, FBLN1, Fibronection and Follistatin were also elevated, suggesting that NFAT1 promotes fibrotic differentiation of mesenchymal cells. To further delineate if NFAT1 has secondary effects on immune cells vs. fibrotic differentiation, immune cells were analyzed by flow cytometry on day 14 post bleomycin injury, and similar number of macrophages, T cells, B cells and dentritic cells were found. Conclusions: NFAT1 deficiency confers protection from lung fibrosis in a bleomycin injury model through dampening the fibrotic activation of lung resident mesenchymal cells. This abstract is funded by NIH grants: RO1F035722, RO1F037975, and T32 training grant (5T32HL007749).