MCT4 Regulates Hypoxia-Mediated Extracellular Lactate Production in IPF Fibroblasts

Abstract Rationale : Idiopathic Pulmonary Fibrosis (IPF) is characterized by a collagen-rich fibrotic extracellular matrix resulting in systemic and microenvironmental hypoxia. Hypoxia promotes extracellular lactate production, and fibroblasts from IPF patients exhibit enhanced lactate levels under hypoxic conditions. We hypothesized that hypoxia-induced lactate production and export from IPF fibroblasts contributes to lung fibrosis. Methods: Normal and IPF fibroblasts (n=5) were cultured under normoxic (21%) or hypoxic (1%) oxygen conditions for 5 days. Intracellular pyruvate, extracellular lactate and expression levels of the lactate transporter proteins, MCT4 (exporter) and MCT1 (importer) were assessed. To investigate lactate's role in myofibroblast differentiation, a-SMA was measured in control fibroblasts that were exposed to conditioned media from IPF fibroblasts cultured in the presence or absence of an MCT4 inhibitor. MCT1, MCT4, and collagen were also assessed in the lungs of C57BL/6 mice (n=4) exposed to hypoxia (10% oxygen) for 21 days and in bleomycin-injured mouse lungs (n=4). Immunohistochemistry was used to assess MCT1 and MCT4 expression in control and IPF lung tissues (n=5 each). Finally, bleomycin-induced lung fibrosis was assessed in C57BL/6 mice treated with an MCT4 inhibitor. Results: Under hypoxic conditions, IPF fibroblasts exhibited decreased intracellular pyruvate and increased extracellular lactate, while pyruvate and lactate were unchanged in control fibroblasts. MCT1 expression was not impacted by hypoxia, while MCT4 was significantly upregulated in IPF, but not control fibroblasts. Silencing MCT4 reduced extracellular lactate levels in IPF fibroblasts under hypoxia. Increased MCT4 expression was observed in IPF lung tissues in hypoxia-exposed murine lungs, and in the bleomycin-treated mice, supporting its role in lung fibrosis. The transfer of conditioned medium from hypoxia-cultured IPF fibroblasts to control fibroblasts increased α-SMA expression, and this was attenuated when the media was from IPF fibroblasts treated with an MCT4 inhibitor. In vivo, treatment with an MCT4 inhibitor significantly reduced collagen deposition in bleomycin-treated mouse lungs. Conclusions: Our findings demonstrate that elevated extracellular lactate in IPF fibroblasts is driven by hypoxia-induced MCT4 expression, and that it may promote the propagation of fibrosis through paracrine intercellular communication. MCT4 inhibition reduced collagen in vivo, indicating that targeting MCT4 may represent a viable therapeutic strategy to mitigate lung fibrosis. We propose a novel paradigm in which lactate export, mediated by dysregulated MCT4, promotes fibrosis in hypoxic microenvironments.