Toll-interacting protein inhibits transforming growth factor beta signaling in mouse lung fibroblasts

Abstract

Variations in the Toll-interacting protein (TOLLIP) gene have been identified in genome-wide association studies to correlate with risk of disease, mortality, and response to N-acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFβ) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFβ and in the bleomycin model of experimental lung fibrosis using Tollip−/− mice. We hypothesize that if TOLLIP negatively regulates TGFβ signaling, then Tollip−/− mouse lung fibroblasts (MLFs) would have enhanced response to TGFβ treatment, and Tollip−/− mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFβ (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA-seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFβ-stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the in vivo model of lung fibrosis, Tollip−/− mice and wild-type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFβ signaling in vivo after bleomycin injury by SMAD2, ERK1/2, and TGFβR1 Western blot. In response to TGFβ treatment, both WT and Tollip−/− MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, Tollip−/− MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of Acta2 by qPCR. Functionally, Tollip−/− MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFβ signaling in Tollip−/− through SMAD2 in vitro and in vivo. Tollip−/− mice experienced lower survival using a standard weight-adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, Tollip−/− mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury.