Toll-like Receptor 9 Inhibition Mitigates Fibroproliferative Responses in Translational Models of Pulmonary Fibrosis

Rationale: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease for which current treatment options only slow clinical progression. Previously, we identified a subset of patients with IPF with an accelerated disease course associated with fibroblast expression of Toll-like receptor 9 (TLR9) mediated by interactions with its ligand mitochondrial DNA. Objectives: We aimed to show that TLR9 activation induces fibroproliferative responses that are abrogated by its antagonism by using two commercially available indirect inhibitors and a proprietary, selective direct small-molecule inhibitor. Methods: We employed two independent cohorts of patients with IPF, multiple in vitro fibroblast cell culture platforms, an in vivo mouse model, and an ex vivo human precision-cut lung slices system to investigate the clinical and biologic significance of TLR9 in this disease. Measurements and Main Results: In two independent IPF cohorts, plasma mitochondrial DNA activates TLR9 in a manner associated with the expression of monocyte chemoattractant protein 1, IL-6, tumor necrosis factor-α, and IFN-γ-inducible protein 10 and worsened transplant-free survival. Our cell culture platform showed that TLR9 mediates fibroblast activation via transforming growth factor-β1 and stiff substrates and that its antagonism, particularly direct inhibition, ameliorates this process, including production of these TLR9-associated pharmacodynamic endpoints. We further demonstrated that direct TLR9 inhibition mitigates these fibroproliferative responses in our in vivo and ex vivo models of pulmonary fibrosis. Conclusions: In this novel study, we found that direct TLR9 inhibition mitigates fibroproliferative responses in preclinical models of pulmonary fibrosis. Our work demonstrates the therapeutic potential of direct TLR9 antagonism in IPF and related fibrotic lung diseases.