Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by extracellular matrix accumulation, which impairs lung compliance and gas exchange. Metabolic profiling of fibrotic lungs shows dysregulation of pathways, including the mitochondrial tricarboxylic acid (TCA) cycle. Cell permeable derivatives of the TCA cycle metabolite succinate (e.g., diethyl succinate or DES) have been developed to directly target mitochondrial metabolism. Studies investigating the therapeutic potential of these compounds in metabolic diseases show that they directly enter cells to exert their effects. Methods The effect of DES was tested on IPF patient-derived lung fibroblasts, normal lung fibroblasts, IPF patient-derived precision cut lung sections (PCLS) and in the bleomycin model of lung fibrosis. In vitro, the effect of DES was assessed using western blots, ELISA, RNAseq, and qPCRs. In mice, lung tissue collagen levels were measured using biochemical and histological assays, and lung function was assessed. Moreover, the effect of DES on the inflammatory tone of the lung was assessed by flow cytometry analysis and cytokine profiling. Results DES treatment of IPF patient fibroblasts reduced type 1 collagen by 68%(p = 0.0007), αSMA by 35%(p = 0.03), and fibronectin by 61%(p = 0.004). DES treatment of TGF-β-activated fibroblasts reduced type 1 collagen by 64%(p = 0.01), and αSMA 30%(p = 0.02). DES reduced the levels of type 1 collagen in IPF patient PCLS by 33%(p = 0.03). RNA sequencing revealed that DES treated fibroblasts downregulated fibrotic fibroblast markers such as CTHRC1 by 54%(p 0.0001) and elevated lipogenic markers such as PLIN2 by 62%(p 0.0001) and PPAR-γ by 29%(p = 0.001). RNA seq analysis was validated using qPCRs and western blots. LipidTOX staining of IPF patient fibroblasts revealed a 10% elevation of lipid droplet accumulation in DES treated fibroblasts (p = 0.0089). In vivo, DES reduced bleomycin-induced elevation of lung tissue collagen levels as assessed by hydroxyproline levels (43% reduction, p = 0.04) and Masson trichrome staining (25% reduction, p = 0.04). DES improved FVC in bleomycin-treated mice by 35%(p = 0.0002). Flow-cytometric analysis revealed that DES dampened fibrosis-associated inflammation in the bleomycin model as seen by the reduction in the numbers of different immune cell populations including inflammatory monocytes, alveolar macrophage subtypes, and dendritic cells among others. Cytokine profiling revealed that DES treatment of bleomycin treated mice led to a specific reduction of lung CCL2 levels (by 51%, p = 0.03), which is crucial for monocyte recruitment to the lung during the progression of lung fibrosis. Conclusion DES exerts anti-fibrotic effects in preclinical models of lung fibrosis, warranting further investigation. This abstract is funded by: Austrian Science Fund (FWF) DOC 129-B
Rajesh et al. (Fri,) studied this question.