Abstract Body (Do not enter title and authors here): Background: Idiopathic Pulmonary fibrosis (IPF) is a complex and deadly disease with limited treatment options. A better understanding of the cellular and molecular mechanisms involved in IPF could contribute to the development of effective therapies for this devastating condition. Among the molecular mediators of interest are the inhibitor of DNA Binding (ID) proteins that modulate critical biological processes including cellular differentiation, proliferation, and cell cycle regulation. However, the regulation and the effects of ID proteins in IPF remain poorly understood.

Methods: Primary human lung fibroblasts were isolated from pulmonary tissue obtained from healthy control donors (NHLF) and patients diagnosed with IPF. Cell proliferation and fibroblast-to-myofibroblast differentiation were evaluated by BrdU incorporation assay and real-time qPCR, respectively. For in vivo experimental modeling of pulmonary fibrosis, WT and ID1/ID3 double knockout mice received a single intratracheal administration of Bleomycin (BLM, 2 U/kg), or sterile saline as control. Animals were euthanized four weeks post-BLM injection, and lung tissues were harvested for subsequent analysis. Pulmonary function test, histopathological and molecular analyses of lung specimens were performed.

Results: ID1 and ID3 mRNA levels were upregulated in human lung fibroblasts treated with TGF-b, a major contributor to lung fibrosis. Moreover, ID1 and ID3 protein levels were upregulated in the lungs of mice and human patients with pulmonary fibrosis. ID1/ID3 inhibition, using a pharmacological inhibitor or specific siRNAs, decreased human lung fibroblasts proliferation and differentiation into myofibroblasts. ID1/ID3 deficient mice exhibited improved lung function and reduced lung fibrosis when exposed to the BLM model. A lung specific inhibition of ID1/ID3, using Adeno-associated Viruses expressing short hairpins targeting ID1 and ID3, effectively reversed pulmonary fibrosis in mice. Mechanistically, we found that ID1/ID3 inhibition decreased fibroblast proliferation through cell cycle genes and inhibited fibroblast differentiation through the MEK/ERK pathway.

Conclusion: Our data demonstrate that both pharmacological inhibition and targeted knockdown of ID1 and ID3 attenuate pulmonary fibrosis. Targeting ID1/ID3 signaling pathway may constitute a novel therapeutic approach for the treatment of IPF.

Keywords: ID proteins, Agx51, Idiopathic Pulmonary fibrosis, Bleomycin.