Human lung fibroblast-to-myofibroblast transformation is not driven by an LDH5-dependent metabolic shift towards aerobic glycolysis

Abstract

Background Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease characterized by aberrant fibroblast activation and progressive fibrotic remodelling of the lungs. Though the exact pathophysiological mechanisms of IPF remain unknown, TGF-β1 is thought to act as a main driver of the disease by mediating fibroblast-to-myofibroblast transformation (FMT). Recent reports have indicated that a metabolic shift towards aerobic glycolysis takes place during FMT and that metabolic shifts can directly influence aberrant cell function. This has led to the hypothesis that inhibition of lactate dehydrogenase 5 (LDH5), an enzyme responsible for converting pyruvate into lactate, could constitute a therapeutic concept for IPF. Methods In this study, we investigated the potential link between aerobic glycolysis and FMT using a potent LDH5 inhibitor (Compound 408, Genentech). Seahorse analysis was performed to determine the effect of Compound 408 on TGF-β1-driven glycolysis in WI-38 fibroblasts. TGF-β1-mediated FMT was measured by quantifying α-smooth muscle actin (α-SMA) and fibronectin in primary human lung fibroblasts following treatment with Compound 408. Lactate and pyruvate levels in the cell culture supernatant were assessed by LC-MS/MS. In addition to pharmacological LDH5 inhibition, the effect of siRNA-mediated knockdown of LDHA and LDHB on FMT was examined. Results We show that treatment of lung fibroblasts with Compound 408 efficiently inhibits LDH5 and attenuates the TGF-β1-mediated metabolic shift towards aerobic glycolysis. Additionally, we demonstrate that LDH5 inhibition has no significant effect on TGF-β1-mediated FMT in primary human lung fibroblasts by analysing α-SMA fibre formation and fibronectin expression. Conclusions Our data strongly suggest that while LDH5 inhibition can prevent metabolic shifts in fibroblasts, it has no influence on FMT and therefore glycolytic dysregulation is unlikely to be the sole driver of FMT. Electronic supplementary material The online version of this article (10.1186/s12931-019-1058-2) contains supplementary material, which is available to authorized users.

AITC inhibits fibroblast-myofibroblast transition via TRPA1-independent MAPK and NRF2/HO-1 pathways and reverses corticosteroids insensitivity in human lung fibroblasts

“…It facilitates pro-inflammatory responses and tissue remodeling in airways [ 15 ]. TGF- β was also identified as the main cytokine that induces augmented matrix deposition mainly through fibroblast recruitment and transformation in the IPF lung [ 16 ]. TGF-β can also stimulate and increase reactive oxygen species (ROS) production that leads oxidant and antioxidant imbalance which in turn can activate latent TGF-β developing a profibrogenic cycle [ 17 ].…”

Real-time measurement of cellular bioenergetics in fully differentiated human nasal epithelial cells grown at air-liquid-interface

“…A range of pathophysiological conditions including cancer and fibrotic lung diseases are thought to involve a reprograming of cellular metabolism, with skewing toward aerobic glycolysis, suggesting a potential therapeutic target. We showed that the addition of a potent LDH5 inhibitor resulted in a dose dependent reduction in extracellular acidification rate, consistent with a reduction in the production of lactate from pyruvate ( 22 ). Our system therefore allowed the evaluation of a novel compound in fully differentiated human cells, with the observed metabolic effects predicted from its designed mode of action.…”

“…This method also has transferability across other disease models where cells are grown on transwell inserts, in particular where there is also a close association between altered glucose metabolism and disease risk ( 5 , 15 , 17 ). In principle this technique would open up additional avenues for metabolic research beyond epithelial monocultures, such as cocultures with fibroblasts which also undergo significant metabolic shifts in disease-relevant conditions ( 22 ). This would support development of novel therapeutic strategies for targeting epithelia-fibroblast crosstalk in idiopathic pulmonary fibrosis and related fibrotic interstitial lung diseases.…”

“…3 ) and injection port D with 65 μl of 25 μM antimycin A and 5 μM Rotenone (final concentrations 2.5 μM and 0.5 μM, respectively). Lactate dehydrogenase 5 (LDH5) inhibitor [synthesized according to the procedures described in patent WO 2015/140133 A1 ( 22 )] was injected to a final concentration of 30 µM per well using DMSO vehicle control where appropriate.…”

Recapitulating idiopathic pulmonary fibrosis related alveolar epithelial dysfunction in a human iPSC‐derived air‐liquid interface model

“…Image analysis was performed as previously described 53,54 . Briefly, using the building blocks of Perkin Elmer´s Columbus Image Analysis system, first nuclei acquired with the Hoechst channel were detected using the building block (BB) “nuclei.” Second, cells were defined with the BB “find cytoplasm” from the FITC channel.…”