Forefront: MiR-34a-Knockout Mice with Wild Type Hematopoietic Cells, Retain Persistent Fibrosis Following Lung Injury.

Raanan Bulvik,Moshe Biton,Raphael Breuer,Shulamit B Wallach-Dayan,Neville Berkman

doi:10.3390/ijms21062228

Abstract

MicroRNAs (miRs) are known to limit gene expression at the post-transcriptional level and have important roles in the pathogenesis of various conditions, including acute lung injury (ALI) and fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). In this study, we found increased levels of miR-34 at times of fibrosis resolution following injury, in myofibroblasts from Bleomycin-treated mouse lungs, which correlates with susceptibility to cell death induced by immune cells. On the contrary, a substantial downregulation of miR-34 was detected at stages of evolution, when fibroblasts resist cell death. Concomitantly, we found an inverse correlation between miR-34 levels with that of the survival molecule FLICE-like inhibitory protein (FLIP) in lung myofibroblasts from humans with IPF and the experimental model. Forced upregulation of miR-34 with miR-34 mimic in human IPF fibrotic-lung myofibroblasts led to decreased cell survival through downregulation of FLIP. Using chimeric miR-34 knock-out (KO)-C57BL/6 mice with miR34KO myofibroblasts but wild-type (WT) hematopoietic cells, we found, in contrast to WT mice, increased and persistent FLIP levels with a more severe fibrosis and with no signs of resolution as detected in pathology and collagen accumulation. Moreover, a mimic of miR-34a decreased FLIP expression and susceptibility to cell death was regained in miR-34KO fibroblasts. Through this study, we show for the first time an inverse correlation between miR-34a and FLIP expression in myofibroblasts, which affects survival, and accumulation in lung fibrosis. Reprogramming fibrotic-lung myofibroblasts to regain susceptibility to cell-death by specifically increasing their miR34a and downregulating FLIP, may be a useful strategy, enabling tissue regeneration following lung injury.

Highlights

Pathological fibrosis is associated with increments in molecules such as FLICE-likeinhibitory-protein (FLIP) allowing escape from immune surveillance and unremitted accumulation of myofibroblasts
In murine lung myofibroblasts accumulating during evolution of fibrosis, that FLIP mediates the deviation from myofibroblast cell-death and apoptosis towards proliferation [1]. miRNA regulation of FLIP, with subsequent resistance to immune-cell induced cell death has not yet been documented in idiopathic pulmonary fibrosis (IPF). miRNAs are a class of evolutionarily conserved non-coding RNAs approximately 20–25 nucleotides in length
FLIP expression increased in human IPF- compared to normal-lung myofibroblasts, from 0.8 ± 0.15 SD (n = 5) to 1.7 ± 0.5 SD (n = 6), respectively (Figure 1A, NL vs. IPF)

Summary

Introduction

Pathological fibrosis is associated with increments in molecules such as FLICE-likeinhibitory-protein (FLIP) allowing escape from immune surveillance and unremitted accumulation of myofibroblasts. In murine lung myofibroblasts accumulating during evolution of fibrosis, that FLIP mediates the deviation from myofibroblast cell-death and apoptosis towards proliferation [1]. In this study, using Bleomycin-induced lung injury and fibrosis in chimeric miR34a-knockout mice (miR34aKO) with miR34aKO mesenchymal cells and wild type (WT) hematopoietic cells, we determined whether disruption of lung tissue-regeneration following injury, with fibrosis evolution, is associated with downregulation of miR34a levels, in myofibroblasts that upregulate FLIP and resist T-cell induced cell-death. We set out to assess whether increasing miR34a levels by a specific mimic affects FLIP expression, and cell-death in IPF-lung myofibroblasts. We determined that miR34a downregulation links FLIP stability with fibroblast capacity to survive and propagate, and that miR34a mimics may regulate evolution of lung fibrosis following injury

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